Methods for inhibiting IL-TIF-induced proliferation of hematopoietic cells using the cytokine receptor Zcytor16

ABSTRACT

Cytokines and their receptors have proven usefulness in both basic research and as therapeutics. The present invention provides a new human cytokine receptor designated as “Zcytor16.”

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/981,998, filed on Nov. 5, 2004, now U.S. Pat. No. 7,189,839, which isa divisional of U.S. application Ser. No. 09/728,911, filed on Dec. 1,2000, now U.S. Pat. No. 6,897,292, which claims the benefit of U.S.Provisional Application Ser. No. 60/169,049, filed on Dec. 3, 1999,Provisional Application Ser. No. 60/232,219, filed on Sep. 13, 2000, andProvisional Application Ser. No. 60/244,610, filed on Oct. 31, 2000, allof which are incorporated herein by reference. Under 35 U.S.C.§119(e)(1), this application claims benefit of said ProvisionalApplications.

TECHNICAL FIELD

The present invention relates generally to a new protein expressed byhuman cells. In particular, the present invention relates to a novelgene that encodes a receptor, designated as “Zcytor16,” and to nucleicacid molecules encoding Zcytor16 polypeptides, and antibodies to thepolypeptide.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the regulation of the growth anddifferentiation of many cell types (see, for example, Arai et al, Annu.Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991);Paul and Seder, Cell 76:241 (1994)). Proteins that constitute thecytokine group include interleukins, interferons, colony stimulatingfactors, tumor necrosis factors, and other regulatory molecules. Forexample, human interleukin-17 is a cytokine which stimulates theexpression of interleukin-6, intracellular adhesion molecule 1,interleukin-8, granulocyte macrophage colony-stimulating factor, andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+ hematopoietic precursors into neutrophils (Yao etal, J. Immunol 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593(1996)).

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that bind the cytokine with high affinity andtransduce this binding event to the cell through the cytoplasmicportions of the certain receptor subunits. Cytokine receptors have beengrouped into several classes on the basis of similarities in theirextracellular ligand binding domains. For example, the receptor chainsresponsible for binding and/or transducing the effect of interferons aremembers of the class II cytokine receptor family, based upon acharacteristic 200 residue extracellular domain.

The demonstrated in vivo activities of cytokines and their receptorsillustrate the clinical potential of, and need for, other cytokines,cytokine receptors, cytokine agonists, and cytokine antagonists.

SUMMARY OF THE INVENTION

The present invention provides a novel receptor, designated “Zcytor16.”The present invention also provides Zcytor16 polypeptides and Zcytor16fusion proteins, as well as nucleic acid molecules encoding suchpolypeptides and proteins, and methods for using these nucleic acidmolecules and amino acid sequences.

DESCRIPTION OF THE INVENTION

1. Overview

An illustrative nucleotide sequence that encodes Zcytor16 is provided bySEQ ID NO:1. The encoded polypeptide has the following amino acidsequence: MMPKHCFLGF LISFFLTGVA GTQSTHESLK PQRVQFQSRN FHNILQWQPGRALTGNSSVY FVQYKIYGQR QWKNKEDCWG TQELSCDLTS ETSDIQEPYY GRVRAASAGSYSEWSMTPRF TPWWETKIDP PVMNITQVNG SLLVILHAPN LPYRYQKEKN VSIEDYYELLYRVFIINNSL EKEQKVYEGA HRAVEIEALT PHSSYCVVAE IYQPMLDRRS QRSEERCVEI P (SEQID NO:2). The 231 amino acid polypeptide represents the extracellulardomain, also called a cytokine-binding domain, of a new class IIcytokine receptor. Features of the Zcytor16 polypeptide include putativesignal sequences at amino acid residues 1 to 21, or 1 to 22, of SEQ IDNO:2, immunoglobulin superfamily (Ig) domains at amino acid residues 22to 108, and 112 to 210, of SEQ ID NO:2, and a linker that residesbetween the Ig domains (i.e., at amino acid residues 109 to 111 of SEQID NO:2). The Zcytor16 gene is expressed in lymphoid, placenta, spleen,tonsil and other tissue, and resides in human chromosome 6q24.1-25.2.

As described below, the present invention provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 80%, orat least 90% identical to a reference amino acid sequence of SEQ ID NO:2selected from the group consisting of: (a) amino acid residues aminoacid residues 21 to 231, (b) amino acid residues 21 to 210, (c) aminoacid residues 22 to 231, (d) amino acid residues 22 to 210, (e) aminoacid residues 22 to 108, (f) amino acid residues 112 to 210, and (g)amino acid residues 21 to 110, wherein the isolated polypeptidespecifically binds with an antibody that specifically binds with apolypeptide consisting of the amino acid sequence of SEQ ID NO:2.Illustrative polypeptides include polypeptides comprising either aminoacid residues 22 to 231 of SEQ ID NO:2 or amino acid residues 22 to 210of SEQ ID NO:2. Moreover, the present invention also provides isolatedpolypeptides as disclosed above that bind IL-TIF (e.g., human IL-TIFpolypeptide sequence as shown in SEQ ID NO:15). The human IL-TIFpolynucleotide sequence is shown in SEQ ID NO:14.

The present invention also provides isolated polypeptides comprising atleast 15 contiguous amino acid residues of an amino acid sequence of SEQID NO:2 selected from the group consisting of: (a) amino acid residuesamino acid residues 21 to 231, (b) amino acid residues 21 to 210, (c)amino acid residues 22 to 231, (d) amino acid residues 22 to 210, (e)amino acid residues 22 to 108, (f) amino acid residues 112 to 210, and(g) amino acid residues 21 to 110. Illustrative polypeptides includepolypeptides that either comprise, or consist of, amino acid residues(a) to (g). Moreover, the present invention also provides isolatedpolypeptides as disclosed above that bind IL-TIF.

The present invention also includes variant Zcytor16 polypeptides,wherein the amino acid sequence of the variant polypeptide shares anidentity with amino acid residues 22 to 210 of SEQ ID NO:2 selected fromthe group consisting of at least 70% identity, at least 80% identity, atleast 90% identity, at least 95% identity, or greater than 95% identity,and wherein any difference between the amino acid sequence of thevariant polypeptide and the corresponding amino acid sequence of SEQ IDNO:2 is due to one or more conservative amino acid substitutions.Moreover, the present invention also provides isolated polypeptides asdisclosed above that bind IL-TIF.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. The presentinvention further includes compositions comprising a carrier and apeptide, polypeptide, or antibody described herein.

The present invention also provides isolated nucleic acid molecules thatencode a Zcytor16 polypeptide, wherein the nucleic acid molecule isselected from the group consisting of: (a) a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:3, (b) a nucleic acidmolecule encoding an amino acid sequence that comprises either aminoacid residues 22 to 231 of SEQ ID NO:2 or amino acid residues 22 to 210of SEQ ID NO:2, and (c) a nucleic acid molecule that remains hybridizedfollowing stringent wash conditions to a nucleic acid moleculecomprising the nucleotide sequence of nucleotides 64 to 630 of SEQ IDNO:1, or the complement of the nucleotide sequence of nucleotides 64 to630 of SEQ ID NO:1. Illustrative nucleic acid molecules include those inwhich any difference between the amino acid sequence encoded by nucleicacid molecule (c) and the corresponding amino acid sequence of SEQ IDNO:2 is due to a conservative amino acid substitution. The presentinvention further contemplates isolated nucleic acid molecules thatcomprise nucleotides 64 to 630 of SEQ ID NO:1. Moreover, the presentinvention also provides isolated polynucleotides that encodepolypeptides as disclosed above that bind IL-TIF.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors maycomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells and recombinant viruses comprising these vectorsand expression vectors. Illustrative host cells include bacterial,yeast, fungal, insect, mammalian, and plant cells. Recombinant hostcells comprising such expression vectors can be used to produce Zcytor16polypeptides by culturing such recombinant host cells that comprise theexpression vector and that produce the Zcytor16 protein, and,optionally, isolating the Zcytor16 protein from the cultured recombinanthost cells.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors. The present invention further includespharmaceutical compositions, comprising a pharmaceutically acceptablecarrier and a polypeptide described herein.

The present invention also contemplates methods for detecting thepresence of Zcytor16 RNA in a biological sample, comprising the steps of(a) contacting a Zcytor16 nucleic acid probe under hybridizingconditions with either (i) test RNA molecules isolated from thebiological sample, or (ii) nucleic acid molecules synthesized from theisolated RNA molecules, wherein the probe has a nucleotide sequencecomprising a portion of the nucleotide sequence of SEQ ID NO:1, or itscomplement, and (b) detecting the formation of hybrids of the nucleicacid probe and either the test RNA molecules or the synthesized nucleicacid molecules, wherein the presence of the hybrids indicates thepresence of Zcytor16 RNA in the biological sample. For example, suitableprobes consist of the following nucleotide sequences of SEQ ID NO:1:nucleotides 64 to 324, nucleotides 64 to 630, nucleotides 334 to 630,and nucleotides 64 to 693. Other suitable probes consist of thecomplement of these nucleotide sequences, or a portion of the nucleotidesequences or their complements.

The present invention further provides methods for detecting thepresence of Zcytor16 polypeptide in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody or anantibody fragment that specifically binds with a polypeptide consistingof the amino acid sequence of SEQ ID NO:2, wherein the contacting isperformed under conditions that allow the binding of the antibody orantibody fragment to the biological sample, and (b) detecting any of thebound antibody or bound antibody fragment. Such an antibody or antibodyfragment may further comprise a detectable label selected from the groupconsisting of radioisotope, fluorescent label, chemiluminescent label,enzyme label, bioluminescent label, and colloidal gold.

The present invention also provides kits for performing these detectionmethods. For example, a kit for detection of Zcytor16 gene expressionmay comprise a container that comprises a nucleic acid molecule, whereinthe nucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule comprising the nucleotide sequence of nucleotides64 to 630 of SEQ ID NO:1, (b) a nucleic acid molecule comprising thecomplement of nucleotides 64 to 630 of the nucleotide sequence of SEQ IDNO:1, (c) a nucleic acid molecule that is a fragment of (a) consistingof at least eight nucleotides, and (d) a nucleic acid molecule that is afragment of (b) consisting of at least eight nucleotides. Such a kit mayalso comprise a second container that comprises one or more reagentscapable of indicating the presence of the nucleic acid molecule. On theother hand, a kit for detection of Zcytor16 protein may comprise acontainer that comprises an antibody, or an antibody fragment, thatspecifically binds with a polypeptide consisting of the amino acidsequence of SEQ ID NO:2.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2. An exemplary anti-idiotypeantibody binds with an antibody that specifically binds a polypeptideconsisting of amino acid residues 22 to 210 of SEQ ID NO:2.

The present invention also provides isolated nucleic acid moleculescomprising a nucleotide sequence that encodes a Zcytor16 secretionsignal sequence and a nucleotide sequence that encodes a biologicallyactive polypeptide, wherein the Zcytor16 secretion signal sequencecomprises an amino acid sequence of residues 1 to 21, of SEQ ID NO:2.Illustrative biologically active polypeptides include Factor VIIa,proinsulin, insulin, follicle stimulating hormone, tissue typeplasminogen activator, tumor necrosis factor, interleukin, colonystimulating factor, interferon, erythropoietin, and thrombopoietin.Moreover, the present invention provides fusion proteins comprising aZcytor16 secretion signal sequence and a polypeptide, wherein theZcytor16 secretion signal sequence comprises an amino acid sequence ofresidues 1 to 21, of SEQ ID NO:2.

The present invention also provides fusion proteins, comprising aZcytor16 polypeptide and an immunoglobulin moiety. In such fusionproteins, the immunoglobulin moiety may be an immunoglobulin heavy chainconstant region, such as a human F_(c) fragment. The present inventionfurther includes isolated nucleic acid molecules that encode such fusionproteins.

The present invention also provides monomeric, homodimeric,heterodimeric and multimeric receptors comprising a zcytor16extracellular domain. Such receptors are soluble or membrane bound, andact as antagonists of the zcytor16 ligand, IL-TIF (e.g., the humanIL-TIF as shown in SEQ ID NO:15). In a preferred embodiment, suchreceptors are soluble receptors comprising at least one zcytor16extracellular domain polypeptide comprising amino acids 22-231, or22-210 of SEQ ID NO:2. The present invention further includes isolatednucleic acid molecules that encode such receptor polypeptides.

The present invention also provides polyclonal and monoclonal antibodiesto monomeric, homodimeric, heterodimeric and multimeric receptorscomprising a zcytor16 extracellular domain such as those describedabove. Moreover, such antibodies can be used antagonize the binding tothe zcytor16 ligand, IL-TIF (SEQ ID NO:15), to the zcytor16 receptor.

The present invention also provides a method for detecting a geneticabnormality in a patient, comprising: obtaining a genetic sample from apatient; producing a first reaction product by incubating the geneticsample with a polynucleotide comprising at least 14 contiguousnucleotides of SEQ ID NO:1 or the complement of SEQ ID NO:1, underconditions wherein said polynucleotide will hybridize to complementarypolynucleotide sequence; visualizing the first reaction product; andcomparing said first reaction product to a control reaction product froma wild type patient, wherein a difference between said first reactionproduct and said control reaction product is indicative of a geneticabnormality in the patient.

The present invention also provides a method for detecting a cancer in apatient, comprising: obtaining a tissue or biological sample from apatient; incubating the tissue or biological sample with an antibody asdescribed above under conditions wherein the antibody binds to itscomplementary polypeptide in the tissue or biological sample;visualizing the antibody bound in the tissue or biological sample; andcomparing levels of antibody bound in the tissue or biological samplefrom the patient to a normal control tissue or biological sample,wherein an increase in the level of antibody bound to the patient tissueor biological sample relative to the normal control tissue or biologicalsample is indicative of a cancer in the patient.

The present invention also provides a method for detecting a cancer in apatient, comprising: obtaining a tissue or biological sample from apatient; labeling a polynucleotide comprising at least 14 contiguousnucleotides of SEQ ID NO:1 or the complement of SEQ ID NO:1; incubatingthe tissue or biological sample with under conditions wherein thepolynucleotide will hybridize to complementary polynucleotide sequence;visualizing the labeled polynucleotide in the tissue or biologicalsample; and comparing the level of labeled polynucleotide hybridizationin the tissue or biological sample from the patient to a normal controltissue or biological sample, wherein an increase in the labeledpolynucleotide hybridization to the patient tissue or biological samplerelative to the normal control tissue or biological sample is indicativeof a cancer in the patient.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al, Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1.47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al, J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that producesZcytor16 from an expression vector. In contrast, Zcytor16 can beproduced by a cell that is a “natural source” of Zcytor16, and thatlacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zcytor16polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities ofZcytor16 using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains,and other linkage to the cell membrane such as via glycophosphoinositol(gpi). Soluble receptors can comprise additional amino acid residues,such as affinity tags that provide for purification of the polypeptideor provide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Solublereceptors can be monomeric, homodimeric, heterodimeric, or multimeric,with multimeric receptors generally not comprising more than 9 subunits,preferably not comprising more than 6 subunits, and most preferably notcomprising more than 3 subunits. Receptor polypeptides are said to besubstantially free of transmembrane and intracellular polypeptidesegments when they lack sufficient portions of these segments to providemembrane anchoring or signal transduction, respectively. Solublereceptors of class I and class II cytokine receptors generally comprisethe extracellular cytokine binding domain free of a transmembrane domainand intracellular domain. For example, representative soluble receptorsinclude a soluble receptor for CRF2-4 (Genbank Accession No. Z17227) asshown in SEQ ID NO:35; a soluble receptor for IL-10R (Genbank AccessionNo.s U00672 and NM_(—)001558) as shown in SEQ ID NO:36; and a solublereceptor for zcytor11 (U.S. Pat. No. 5,965,704) as shown in SEQ IDNO:34. It is well within the level of one of skill in the art todelineate what sequences of a known class I or class II cytokinesequence comprise the extracellular cytokine binding domain free of atransmembrane domain and intracellular domain. Moreover, one of skill inthe art using the genetic code can readily determine polynucleotidesthat encode such soluble receptor polypeptides.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of ananti-Zcytor16 antibody, and thus, an anti-idiotype antibody mimics anepitope of Zcytor16.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-Zcytor16 monoclonal antibody fragmentbinds with an epitope of Zcytor16.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al, EMBO J. 4:1075 (1985);Nilsson et al, Methods Enzymol 198:3 (1991)), glutathione S transferase(Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al, Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and a Zcytor16polypeptide component. Examples of an antibody fusion protein include aprotein that comprises a Zcytor16 extracellular domain, and either an Fcdomain or an antigen-biding region.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Zcytor16” or a “Zcytor16anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the Zcytor16 gene,or (b) capable of forming a stable duplex with a portion of an mRNAtranscript of the Zcytor16 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant Zcytor16 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of Zcytor16 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of Zcytor16 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant Zcytor16 gene can be identified,for example, by determining whether the gene hybridizes with a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, or itscomplement, under stringent conditions.

Alternatively, variant Zcytor16 genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant Zcytor16gene or variant Zcytor16 polypeptide, a variant gene or polypeptideencoded by a variant gene may be functionally characterized the abilityto bind specifically to an anti-Zcytor16 antibody. A variant Zcytor16gene or variant Zcytor16 polypeptide may also be functionallycharacterized the ability to bind to its ligand, IL-TIF, using abiological or biochemical assay described herein.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of specification.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of Zcytor16 genes.Within the context of this invention, a “functional fragment” of aZcytor16 gene refers to a nucleic acid molecule that encodes a portionof a Zcytor16 polypeptide which is a domain described herein or at leastspecifically binds with an anti-Zcytor16 antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of Zcytor16 Polynucleotides or Genes

Nucleic acid molecules encoding a human Zcytor16 gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1. These techniques are standard andwell-established.

As an illustration, a nucleic acid molecule that encodes a humanZcytor16 gene can be isolated from a cDNA library. In this case, thefirst step would be to prepare the cDNA library by isolating RNA from atissue, such as tonsil tissue, using methods well-known to those ofskill in the art. In general, RNA isolation techniques must provide amethod for breaking cells, a means of inhibiting RNase-directeddegradation of RNA, and a method of separating RNA from DNA, protein,and polysaccharide contaminants. For example, total RNA can be isolatedby freezing tissue in liquid nitrogen, grinding the frozen tissue with amortar and pestle to lyse the cells, extracting the ground tissue with asolution of phenol/chloroform to remove proteins, and separating RNAfrom the remaining impurities by selective precipitation with lithiumchloride (see, for example, Ausubel et al (eds.), Short Protocols inMolecular Biology, 3^(rd) Edition, pages 4-1 to 4-6 (John Wiley & Sons1995) [“Ausubel (1995)”]; Wu et al, Methods in Gene Biotechnology, pages33-41 (CRC Press, Inc. 1997) [“Wu (1997)”]).

Alternatively, total RNA can be isolated by extracting ground tissuewith guanidinium isothiocyanate, extracting with organic solvents, andseparating RNA from contaminants using differential centrifugation (see,for example, Chirgwin et al, Biochemistry 18:52 (1979); Ausubel (1995)at pages 4-1 to 4-6; Wu (1997) at pages 33-41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol I, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM-4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned cDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 or DH10B cells,which can be obtained, for example, from Life Technologies, Inc. orGIBCO BRL (Gaithersburg, Md.).

A human genomic library can be prepared by means well known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Manassas, Va.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO:1, using standardmethods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

Nucleic acid molecules that encode a human Zcytor16 gene can also beobtained using the polymerase chain reaction (PCR) with oligonucleotideprimers having nucleotide sequences that are based upon the nucleotidesequences of the Zcytor16 gene, as described herein. General methods forscreening libraries with PCR are provided by, for example, Yu et al,“Use of the Polymerase Chain Reaction to Screen Phage Libraries,” inMethods in Molecular Biology, Vol. 15. PCR Protocols: Current Methodsand Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).Moreover, techniques for using PCR to isolate related genes aredescribed by, for example, Preston, “Use of Degenerate OligonucleotidePrimers and the Polymerase Chain Reaction to Clone Gene Family Members,”in Methods in Molecular Biology, Vol 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 317-337 (Humana Press, Inc. 1993).

Anti-Zcytor16 antibodies, produced as described below, can also be usedto isolate DNA sequences that encode human Zcytor16 genes from cDNAlibraries. For example, the antibodies can be used to screen λgt11expression libraries, or the antibodies can be used for immunoscreeningfollowing hybrid selection and translation (see, for example, Ausubel(1995) at pages 6-12 to 6-16; Margolis et al., “Screening λ expressionlibraries with antibody and protein probes,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al (eds.), pages 1-14 (OxfordUniversity Press 1995)).

As an alternative, a Zcytor16 gene can be obtained by synthesizingnucleic acid molecules using mutually priming long oligonucleotides andthe nucleotide sequences described herein (see, for example, Ausubel(1995) at pages 8-8 to 8-9). Established techniques using the polymerasechain reaction provide the ability to synthesize DNA molecules at leasttwo kilobases in length (Adang et al., Plant Molec. Biol 21:1131 (1993),Bambot et al., PCR Methods and Applications 2:266 (1993), Dillon et al.,“Use of the Polymerase Chain Reaction for the Rapid Construction ofSynthetic Genes,” in Methods in Molecular Biology, Vol 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993), and Holowachuketal, PCR Methods Appl 4:299(1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al, Annu. Rev. Biochem. 53:323 (1984), andClimie et al, Proc. Nat'l Acad. Sci. USA 87:633 (1990).

The sequence of a Zcytor16 cDNA or Zcytor16 genomic fragment can bedetermined using standard methods. Zcytor16 polynucleotide sequencesdisclosed herein can also be used as probes or primers to clone 5′non-coding regions of a Zcytor16 gene. Promoter elements from a Zcytor16gene can be used to direct the expression of heterologous genes in, forexample, tonsil tissue of transgenic animals or patients treated withgene therapy. The identification of genomic fragments containing aZcytor16 promoter or regulatory element can be achieved usingwell-established techniques, such as deletion analysis (see, generally,Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of Zcytor16proteins by “gene activation,” as disclosed in U.S. Pat. No. 5,641,670.Briefly, expression of an endogenous Zcytor16 gene in a cell is alteredby introducing into the Zcytor16 locus a DNA construct comprising atleast a targeting sequence, a regulatory sequence, an exon, and anunpaired splice donor site. The targeting sequence is a Zcytor16 5′non-coding sequence that permits homologous recombination of theconstruct with the endogenous Zcytor16 locus, whereby the sequenceswithin the construct become operably linked with the endogenous Zcytor16coding sequence. In this way, an endogenous Zcytor16 promoter can bereplaced or supplemented with other regulatory sequences to provideenhanced, tissue-specific, or otherwise regulated expression.

4. Production of Zcytor16 Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, that encode the Zcytor16 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate nucleotide sequence that encompasses all nucleic acidmolecules that encode the Zcytor16 polypeptide of SEQ ID NO:2. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:3 also provides all RNA sequences encoding SEQ ID NO:2, bysubstituting U for T. Moreover, the present invention also providesisolated soluble monomeric, homodimeric, heterodimeric and multimericreceptor polypeptides that comprise at least one zcytor16 receptorsubunit that is substantially homologous to the receptor polypeptide ofSEQ ID NO:3. Thus, the present invention contemplates Zcytor16polypeptide-encoding nucleic acid molecules comprising nucleotide 1 tonucleotide 693 of SEQ ID NO:1, and their RNA equivalents.

Table 1 sets forth the one-letter codes used within SEQ ID NO:3 todenote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter • TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequences of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al, Nucl Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al, Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed herein serve as a template foroptimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zcytor16 polypeptidesfrom other mammalian species, including mouse, porcine, ovine, bovine,canine, feline, equine, and other primate polypeptides. Orthologs ofhuman Zcytor16 can be cloned using information and compositions providedby the present invention in combination with conventional cloningtechniques. For example, a Zcytor16 cDNA can be cloned using mRNAobtained from a tissue or cell type that expresses Zcytor16 as disclosedherein. Suitable sources of mRNA can be identified by probing northernblots with probes designed from the sequences disclosed herein. Alibrary is then prepared from mRNA of a positive tissue or cell line.

A Zcytor16-encoding cDNA can be isolated by a variety of methods, suchas by probing with a complete or partial human cDNA or with one or moresets of degenerate probes based on the disclosed sequences. A cDNA canalso be cloned using the polymerase chain reaction with primers designedfrom the representative human Zcytor16 sequences disclosed herein. Inaddition, a cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to Zcytor16 polypeptide.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human Zcytor16, and thatallelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the nucleotide sequences disclosedherein, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention, as are proteins which are allelic variants of theamino acid sequences disclosed herein. cDNA molecules generated fromalternatively spliced mRNAs, which retain the properties of the Zcytor16polypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that comprise a soluble receptorsubunit that is substantially homologous to SEQ ID NO:1 or SEQ ID NO:2,SEQ ID NO:13, amino acids 22-210 of SEQ ID NO:2, or allelic variantsthereof and retain the ligand-binding properties of the wild-typezcytor16 receptor. Such polypeptides may also include additionalpolypeptide segments as generally disclosed herein.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules comprising the nucleotide sequence of SEQ IDNO:1, to nucleic acid molecules consisting of the nucleotide sequence ofnucleotides 64 to 693 of SEQ ID NO:1, or to nucleic acid moleculescomprising a nucleotide sequence complementary to SEQ ID NO:1 or tonucleotides 64 to 693 of SEQ ID NO:1. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polynucleotide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions which influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). Typically, hybridization buffers containfrom between 10 mM-1 M Na⁺. The addition of destabilizing or denaturingagents such as formamide, tetralkylammonium salts, guanidinium cationsor thiocyanate cations to the hybridization solution will alter theT_(m) of a hybrid. Typically, formamide is used at a concentration of upto 50% to allow incubations to be carried out at more convenient andlower temperatures. Formamide also acts to reduce non-specificbackground when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant Zcytor16polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC, 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution (100× Denhardt's solution: 2%(w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovineserum albumin), 10% dextran sulfate, and 20 μg/ml denatured, shearedsalmon sperm DNA. One of skill in the art can devise variations of thesehybridization conditions. For example, the hybridization mixture can beincubated at a higher temperature, such as about 65° C., in a solutionthat does not contain formamide. Moreover, premixed hybridizationsolutions are available (e.g., EXPRESSHYB Hybridization Solution fromCLONTECH Laboratories, Inc.), and hybridization can be performedaccording to the manufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. As an illustration, nucleic acidmolecules encoding a variant Zcytor16 polypeptide remain hybridized witha nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1(or its complement) under stringent washing conditions, in which thewash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. For example, nucleic acid molecules encoding a variantZcytor16 polypeptide remain hybridized with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1 (or its complement) underhighly stringent washing conditions, in which the wash stringency isequivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSCwith 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated Zcytor16 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having at least70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the sequences shown in SEQ ID NO:2, or theirorthologs.

The present invention also contemplates Zcytor16 variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and a hybridization assay, as described above.Such Zcytor16 variants include nucleic acid molecules (1) that remainhybridized with a nucleic acid molecule having the nucleotide sequenceof SEQ ID NO:1 (or its complement) under stringent washing conditions,in which the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDSat 55-65° C., and (2) that encode a polypeptide having at least 70%, atleast 80%, at least 90%, at least 95% or greater than 95% sequenceidentity to the amino acid sequence of SEQ ID NO:2. Alternatively,Zcytor16 variants can be characterized as nucleic acid molecules (1)that remain hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) that encode apolypeptide having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the amino acid sequence ofSEQ ID NO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100).

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZcytor16 variant. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:2) and a test sequence that have either the highest density ofidentities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with anamino acid sequence disclosed herein. For example, variants can beobtained that contain one or more amino acid substitutions of SEQ IDNO:2, in which an alkyl amino acid is substituted for an alkyl aminoacid in a Zcytor16 amino acid sequence, an aromatic amino acid issubstituted for an aromatic amino acid in a Zcytor16 amino acidsequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a Zcytor16 amino acid sequence, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a Zcytor16 amino acid sequence, an acidic amino acid issubstituted for an acidic amino acid in a Zcytor16 amino acid sequence,a basic amino acid is substituted for a basic amino acid in a Zcytor16amino acid sequence, or a dibasic monocarboxylic amino acid issubstituted for a dibasic monocarboxylic amino acid in a Zcytor16 aminoacid sequence. Among the common amino acids, for example, a“conservative amino acid substitution” is illustrated by a substitutionamong amino acids within each of the following groups: (1) glycine,alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine,and tryptophan, (3) serine and threonine, (4) aspartate and glutamate,(5) glutamine and asparagine, and (6) lysine, arginine and histidine.The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Particular variants of Zcytor16 are characterized by having at least70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the corresponding amino acid sequence (e.g., SEQ IDNO:2), wherein the variation in amino acid sequence is due to one ormore conservative amino acid substitutions.

Conservative amino acid changes in a Zcytor16 gene can be introduced,for example, by substituting nucleotides for the nucleotides recited inSEQ ID NO:1. Such “conservative amino acid” variants can be obtained byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), DirectedMutagenesis: A Practical Approach (IRL Press 1991)). A variant Zcytor16polypeptide can be identified by the ability to specifically bindanti-Zcytor16 antibodies.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al, J. Am. Chem. Soc.113:2722 (1991), Ellman et al, Methods Enzymol 202:301 (1991), Chung etal, Science 259:806 (1993), and Chung et al, Proc. Nat'l Acad. Sci. USA90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al, J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al, Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for Zcytor16 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al, Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al, J. Biol. Chem.271:4699 (1996).

Although sequence analysis can be used to further define the Zcytor16ligand binding region, amino acids that play a role in Zcytor16 bindingactivity (such as binding of zcytor16 to ligand IL-TIF, or to ananti-zcytor16 antibody) can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., Science 255:306 (1992), Smith etal, J. Mol. Biol 224:899 (1992), and Wlodaver et al, FEBS Lett. 309:59(1992).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal, U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al, Gene 46:145(1986), and Ner et al, DNA 7:127, (1988)). Moreover, Zcytor16 labeledwith biotin or FITC can be used for expression cloning of Zcytor16ligands.

Variants of the disclosed Zcytor16 nucleotide and polypeptide sequencescan also be generated through DNA shuffling as disclosed by Stemmer,Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747(1994), and international publication No. WO 97/20078. Briefly, variantDNA molecules are generated by in vitro homologous recombination byrandom fragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zcytor16 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of Zcytor16polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a Zcytor16 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for the ability to bindanti-Zcytor16 antibodies. One alternative to exonuclease digestion is touse oligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a Zcytor16 gene can be synthesized using thepolymerase chain reaction.

This general approach is exemplified by studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al, Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol 1, Boynton etal, (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al, J.Biol. Chem. 270:29270 (1995); Fukunaga et al, J. Biol. Chem. 270:25291(1995); Yamaguchi et al, Biochem. Pharmacol 50:1295 (1995), and Meiselet al, Plant Molec. Biol 30:1 (1996).

Analysis of the particular sequences disclosed herein provide a set ofillustrative functional fragments presented in Table 4.

TABLE 4 Amino acid residues Nucleotides Zcytor16 Feature (SEQ ID NO: 2)(SEQ ID NO: 1) First Ig Domain 22-108 64-324 Second Ig Domain 112-210 334-630  Both Ig Domains 22-210 64-630

The present invention also contemplates functional fragments of aZcytor16 gene that have amino acid changes, compared with an amino acidsequence disclosed herein. A variant Zcytor16 gene can be identified onthe basis of structure by determining the level of identity withdisclosed nucleotide and amino acid sequences, as discussed above. Analternative approach to identifying a variant gene on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant Zcytor16 gene can hybridize to a nucleic acid moleculecomprising a nucleotide sequence, such as SEQ ID NO:1.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a Zcytor16 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al, Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al, Science 219:660 (1983)).Accordingly, antigenic epitope-bearing peptides and polypeptides of thepresent invention are useful to raise antibodies that bind with thepolypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of an amino acid sequence disclosed herein.Such epitope-bearing peptides and polypeptides can be produced byfragmenting a Zcytor16 polypeptide, or by chemical peptide synthesis, asdescribed herein. Moreover, epitopes can be selected by phage display ofrandom peptide libraries (see, for example, Lane and Stephen, Curr.Opin. Immunol 5:268 (1993), and Cortese et al, Curr. Opin. Biotechnol7:616 (1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992), Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

For any Zcytor16 polypeptide, including variants and fusion proteins,one of ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise Zcytor16 variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of the following sequences:SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Suitable forms ofcomputer-readable media include magnetic media and optically-readablemedia. Examples of magnetic media include a hard or fixed drive, arandom access memory (RAM) chip, a floppy disk, digital linear tape(DLT), a disk cache, and a ZIP disk. Optically readable media areexemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CD-recordable), and digital versatile/videodiscs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

6. Production of Zcytor16 Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides; soluble monomeric, homodimeric, heterodimeric andmultimeric receptors; full-length receptors; receptor fragments (e.g.ligand-binding fragments), functional fragments, and fusion proteins,can be produced in recombinant host cells following conventionaltechniques. To express a Zcytor16 gene, a nucleic acid molecule encodingthe polypeptide must be operably linked to regulatory sequences thatcontrol transcriptional expression in an expression vector and then,introduced into a host cell. In addition to transcriptional regulatorysequences, such as promoters and enhancers, expression vectors caninclude translational regulatory sequences and a marker gene which issuitable for selection of cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a Zcytor16 expression vector maycomprise a Zcytor16 gene and a secretory sequence derived from anysecreted gene.

Zcytor16 proteins of the present invention may be expressed in mammaliancells. Examples of suitable mammalian host cells include African greenmonkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin etal., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1;ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and murine embryonic cells (1H-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al, Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control Zcytor16 gene expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al, Mol Cell Biol 10:4529 (1990), and Kaufman et al,Nucl Acids Res. 19:4485 (1991)).

In certain embodiments, a DNA sequence encoding a Zcytor16 monomeric orhomodimeric soluble receptor polypeptide, or a DNA sequence encoding anadditional subunit of a heterodimeric or multimeric Zcytor16 solublereceptor, e.g., CRF2-4 or IL10R, polypeptide is operably linked to othergenetic elements required for its expression, generally including atranscription promoter and terminator, within an expression vector. Thevector will also commonly contain one or more selectable markers and oneor more origins of replication, although those skilled in the art willrecognize that within certain systems selectable markers may be providedon separate vectors, and replication of the exogenous DNA may beprovided by integration into the host cell genome. Selection ofpromoters, terminators, selectable markers, vectors and other elementsis a matter of routine design within the level of ordinary skill in theart. Many such elements are described in the literature and areavailable through commercial suppliers. Multiple components of a solublereceptor complex can be co-transfected on individual expression vectorsor be contained in a single expression vector. Such techniques ofexpressing multiple components of protein complexes are well known inthe art.

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Zcytor16 polypeptides can also be produced by cultured mammalian cellsusing a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, retroviruses, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see Becker et al, Meth. CellBiol 43:161 (1994), and Douglas and Curiel, Science & Medicine 4.44(1997)). Advantages of the adenovirus system include the accommodationof relatively large DNA inserts, the ability to grow to high-titer, theability to infect a broad range of mammalian cell types, and flexibilitythat allows use with a large number of available vectors containingdifferent promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al, Cytotechnol 15:145 (1994)).

Zcytor16 can also be expressed in other higher eukaryotic cells, such asavian, fungal, insect, yeast, or plant cells. The baculovirus systemprovides an efficient means to introduce cloned Zcytor16 genes intoinsect cells. Suitable expression vectors are based upon the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV), and containwell-known promoters such as Drosophila heat shock protein (hsp) 70promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the Zcytor16 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed Zcytor16 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al, Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a Zcytor16 gene is transformed into E. coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol71:971 (1990), Bonning, et al, J. Gen. Virol 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native Zcytor16 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native Zcytor16 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf-21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al, “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al, “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanotica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al, U.S. Pat. No. 4,845,075. Transformed cells are selected byphenotype determined by the selectable marker, commonly drug resistanceor the ability to grow in the absence of a particular nutrient (e.g.,leucine). A suitable vector system for use in Saccharomyces cerevisiaeis the POT1 vector system disclosed by Kawasaki et al (U.S. Pat. No.4,931,373), which allows transformed cells to be selected by growth inglucose-containing media. Additional suitable promoters and terminatorsfor use in yeast include those from glycolytic enzyme genes (see, e.g.,Kawasaki, U.S. Pat. No. 4,599,311, Kingsman et al, U.S. Pat. No.4,615,974, and Bitter, U.S. Pat. No. 4,977,092) and alcoholdehydrogenase genes. See also U.S. Pat. Nos. 4,990,446, 5,063,154,5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, it is preferred to have the entireexpression segment of the plasmid flanked at both ends by host DNAsequences. A suitable selectable marker for use in Pichia methanolica isa P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be deficient in vacuolar proteasegenes (PEP4 and PRB1). Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al, Science 227:1229 (1985), Kleinet al, Biotechnology 10:268 (1992), and Miki et al, “Procedures forIntroducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al (eds.), pages 67-88 (CRC Press,1993).

Alternatively, Zcytor16 genes can be expressed in prokaryotic hostcells. Suitable promoters that can be used to express Zcytor16polypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the P_(R) and P_(L) promoters of bacteriophage lambda,the trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZpromoters of E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloram-phenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal, Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al (1995).

Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH41, DH5, DH51, DH51F′, DH51MCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a Zcytor16 polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al, “Expressionof foreign proteins in E. coli using plasmid vectors and purification ofspecific polyclonal antibodies,” in DNA Cloning 2: Expression Systems,2nd Edition, Glover et al (eds.), page 15 (Oxford University Press1995), Ward et al, “Genetic Manipulation and Expression of Antibodies,”in Monoclonal Antibodies: Principles and Applications, page 137(Wiley-Liss, Inc. 1995), and Georgiou, “Expression of Proteins inBacteria,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), page 101 (John Wiley & Sons, Inc. 1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al, Science 266:776 (1994),Hackeng et al, Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NO:2. As an illustration, polypeptides can comprise at least six, atleast nine, or at least 15 contiguous amino acid residues of any of thefollowing amino acid sequences of SEQ ID NO:2: amino acid residues aminoacid residues 21 to 231, amino acid residues 21 to 210, amino acidresidues 22 to 231, amino acid residues 22 to 210, amino acid residues22 to 108, amino acid residues 112 to 210, and amino acid residues 21 to110. Within certain embodiments of the invention, the polypeptidescomprise 20, 30, 40, 50, 100, or more contiguous residues of these aminoacid sequences. Nucleic acid molecules encoding such peptides andpolypeptides are useful as polymerase chain reaction primers and probes.

Moreover, zcytor16 polypeptides can be expressed as monomers,homodimers, heterodimers, or multimers within higher eukaryotic cells.Such cells can be used to produce zcytor16 monomeric, homodimeric,heterodimeric and multimeric receptor polypeptides that comprise atleast one zcytor16 polypeptide (“zcytor16-comprising receptors” or“zcytor16-comprising receptor polypeptides”), or can be used as assaycells in screening systems. Within one aspect of the present invention,a polypeptide of the present invention comprising the zcytor16extracellular domain is produced by a cultured cell, and the cell isused to screen for ligands for the receptor, including the naturalligand, IL-TIF, as well as agonists and antagonists of the naturalligand. To summarize this approach, a cDNA or gene encoding the receptoris combined with other genetic elements required for its expression(e.g., a transcription promoter), and the resulting expression vector isinserted into a host cell. Cells that express the DNA and producefunctional receptor are selected and used within a variety of screeningsystems. Each component of the monomeric, homodimeric, heterodimeric andmultimeric receptor complex can be expressed in the same cell. Moreover,the components of the monomeric, homodimeric, heterodimeric andmultimeric receptor complex can also be fused to a transmembrane domainor other membrane fusion moiety to allow complex assembly and screeningof transfectants as described above.

Mammalian cells suitable for use in expressing Zcytor16 receptors andtransducing a receptor-mediated signal include cells that express otherreceptor subunits that may form a functional complex with Zcytor16.These subunits may include those of the interferon receptor family or ofother class II or class I cytokine receptors, e.g., CRF2-4 (GenbankAccession No. Z17227), IL-10R (Genbank Accession No.s U00672 andNM_(—)001558), zcytor11 (U.S. Pat. No. 5,965,704), zcytor7 (U.S. Pat.No. 5,945,511), and IL-9R. It is also preferred to use a cell from thesame species as the receptor to be expressed. Within a preferredembodiment, the cell is dependent upon an exogenously suppliedhematopoietic growth factor for its proliferation. Preferred cell linesof this type are the human TF-1 cell line (ATCC number CRL-2003) and theAML-193 cell line (ATCC number CRL-9589), which are GM-CSF-dependenthuman leukemic cell lines and BaF3 (Palacios and Steinmetz, Cell 41:727-734, (1985)) which is an IL-3 dependent murine pre-B cell line.Other cell lines include BHK, COS-1 and CHO cells.

Suitable host cells can be engineered to produce the necessary receptorsubunits or other cellular component needed for the desired cellularresponse. This approach is advantageous because cell lines can beengineered to express receptor subunits from any species, therebyovercoming potential limitations arising from species specificity.Species orthologs of the human receptor cDNA can be cloned and usedwithin cell lines from the same species, such as a mouse cDNA in theBaF3 cell line. Cell lines that are dependent upon one hematopoieticgrowth factor, such as GM-CSF or IL-3, can thus be engineered to becomedependent upon another cytokine that acts through the zcytor16 receptor,such as IL-TIF.

Cells expressing functional receptor are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in a target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the metabolic breakdown of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assayformat uses cells that are further engineered to express a reportergene. The reporter gene is linked to a promoter element that isresponsive to the receptor-linked pathway, and the assay detectsactivation of transcription of the reporter gene. A preferred promoterelement in this regard is a serum response element, or SRE. See, e.g.,Shaw et al, Cell 56:563-572, (1989). A preferred such reporter gene is aluciferase gene (de Wet et al., Mol Cell Biol. 7:725, (1987)).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al, J. Biol. Chem.269:29094-29101, (1994); Schenborn and Goiffin, Promega Notes 41:11,1993). Luciferase activity assay kits are commercially available from,for example, Promega Corp., Madison, Wis. Target cell lines of this typecan be used to screen libraries of chemicals, cell-conditioned culturemedia, fungal broths, soil samples, water samples, and the like. Forexample, a bank of cell-conditioned media samples can be assayed on atarget cell to identify cells that produce ligand. Positive cells arethen used to produce a cDNA library in a mammalian expression vector,which is divided into pools, transfected into host cells, and expressed.Media samples from the transfected cells are then assayed, withsubsequent division of pools, re-transfection, subculturing, andre-assay of positive cells to isolate a cloned cDNA encoding the ligand.

A natural ligand for the Zcytor16 receptor can also be identified bymutagenizing a cell line expressing the full-length receptor or receptorfusion (e.g., comprising the zcytor16 extracellular domain fused to thetransmembrane and signaling domain of another cytokine receptor) andculturing it under conditions that select for autocrine growth. See WIPOpublication WO 95/21930. Within a typical procedure, IL-3 dependent BaF3cells expressing Zcytor16 and the necessary additional subunits aremutagenized, such as with 2-ethylmethanesulfonate (EMS). The cells arethen allowed to recover in the presence of IL-3, then transferred to aculture medium lacking IL-3 and IL-4. Surviving cells are screened forthe production of a zcytor16 ligand (e.g., IL-TIF), such as by addingsoluble receptor to the culture medium or by assaying conditioned mediaon wild-type BaF3 cells and BaF3 cells expressing the receptor. Usingthis method, cells and tissues expressing IL-TIF can be identified.

Moreover several IL-TIF responsive cell lines are known (Dumontier etal., J. Immunol. 164:1814-1819, 2000; Dumoutier, L. et al., Proc. Nat'l.Acad. Sci. 97:10144-10149, 2000; Xie M H et al., J. Biol. Chem. 275:31335-31339, 2000; Kotenko S V et al., JBC in press), as well as thosethat express the IL-TIF receptor subunit zcytor11. For example, thefollowing cells are responsive to IL-TIF: TK-10 (Xie M H et al., supra.)(human renal carcinoma); SW480 (ATCC No. CCL-228) (human colonadenocarcinoma); HepG2 (ATCC No. HB-8065) (human hepatoma); PC12 (ATCCNo. CRL-1721) (murine neuronal cell model; rat pheochromocytoma); andMES13 (ATCC No. CRL-1927) (murine kidney mesangial cell line). Inaddition, some cell lines express zcytor11 (IL-TIF receptor) are alsocandidates for responsive cell lines to IL-TIF: A549 (ATCC No. CCL-185)(human lung carcinoma); G-361 (ATCC No. CRL-1424) (human melanoma); andCaki-1 (ATCC No. HTB-46) (human renal carcinoma). These cells can beused in assays to assess the functionality of zcytor16 as an IL-TIFantagonist or anti-inflammatory factor.

An additional screening approach provided by the present inventionincludes the use of hybrid receptor polypeptides. These hybridpolypeptides fall into two general classes. Within the first class, theintracellular domain of Zcytor16, is joined to the ligand-binding domainof a second receptor. It is preferred that the second receptor be ahematopoietic cytokine receptor, such as mpl receptor (Souyri et al,Cell 63: 1137-1147, (1990). The hybrid receptor will further comprise atransmembrane domain, which may be derived from either receptor. A DNAconstruct encoding the hybrid receptor is then inserted into a hostcell. Cells expressing the hybrid receptor are cultured in the presenceof a ligand for the binding domain (e.g., TPO in the case the mplreceptor extracellular domain is used) and assayed for a response. Thissystem provides a means for analyzing signal transduction mediated byZcytor16 while using readily available ligands. This system can also beused to determine if particular cell lines are capable of responding tosignals transduced by Zcytor16 monomeric, homodimeric, heterodimeric andmultimeric receptors of the present invention.

A second class of hybrid receptor polypeptides comprise theextracellular (ligand-binding) domain of Zcytor16 (approximatelyresidues 22 to 231 of SEQ ID NO:2; SEQ ID NO:13) with an intracellulardomain of a second receptor, preferably a hematopoietic cytokinereceptor, and a transmembrane domain. Hybrid zcytor11 monomers,homodimers, heterodimers and multimers of the present inventionreceptors of this second class are expressed in cells known to becapable of responding to signals transduced by the second receptor.Together, these two classes of hybrid receptors enable theidentification of a responsive cell type for the development of an assayfor detecting IL-TIF. Moreover, such cells can be used in the presenceof IL-TIF to assay the soluble receptor antagonists of the presentinvention in a competition-type assay. In such assay, a decrease in theproliferation or signal transduction activity of IL-TIF in the presenceof a soluble receptor of the present invention demonstrates antagonisticactivity. Moreover IL-TIF-soluble receptor binding assays can also beused to assess whether a soluble receptor antagonizes IL-TIF activity.

7. Production of Zcytor16 Fusion Proteins and Conjugates

One general class of Zcytor16 analogs are variants having an amino acidsequence that is a mutation of the amino acid sequence disclosed herein.Another general class of Zcytor16 analogs is provided by anti-idiotypeantibodies, and fragments thereof, as described below. Moreover,recombinant antibodies comprising anti-idiotype variable domains can beused as analogs (see, for example, Monfardini et al, Proc. Assoc. Am.Physicians 108:420 (1996)). Since the variable domains of anti-idiotypeZcytor16 antibodies mimic Zcytor16, these domains can provide Zcytor16binding activity. Methods of producing anti-idiotypic catalyticantibodies are known to those of skill in the art (see, for example,Joron et al, Ann. N Y Acad. Sci. 672:216 (1992), Friboulet et al, ApplBiochem. Biotechnol 47:229 (1994), and Avalle et al, Ann. N Y Acad. Sci.864:118 (1998)).

Another approach to identifying Zcytor16 analogs is provided by the useof combinatorial libraries. Methods for constructing and screening phagedisplay and other combinatorial libraries are provided, for example, byKay et al, Phage Display of Peptides and Proteins (Academic Press 1996),Verdine, U.S. Pat. No. 5,783,384, Kay, et. al, U.S. Pat. No. 5,747,334,and Kauffman et al, U.S. Pat. No. 5,723,323.

Zcytor16 polypeptides have both in vivo and in vitro uses. As anillustration, a soluble form of Zcytor16 can be added to cell culturemedium to inhibit the effects of the Zcytor16 ligand produced by thecultured cells.

Fusion proteins of Zcytor16 can be used to express Zcytor16 in arecombinant host, and to isolate the produced Zcytor16. As describedbelow, particular Zcytor16 fusion proteins also have uses in diagnosisand therapy. One type of fusion protein comprises a peptide that guidesa Zcytor16 polypeptide from a recombinant host cell. To direct aZcytor16 polypeptide into the secretory pathway of a eukaryotic hostcell, a secretory signal sequence (also known as a signal peptide, aleader sequence, prepro sequence or pre sequence) is provided in theZcytor16 expression vector. While the secretory signal sequence may bederived from Zcytor16, a suitable signal sequence may also be derivedfrom another secreted protein or synthesized de novo. The secretorysignal sequence is operably linked to a Zcytor16-encoding sequence suchthat the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the nucleotide sequence encoding thepolypeptide of interest, although certain secretory signal sequences maybe positioned elsewhere in the nucleotide sequence of interest (see,e.g., Welch et al, U.S. Pat. No. 5,037,743; Holland et al, U.S. Pat. No.5,143,830).

Although the secretory signal sequence of Zcytor16 or another proteinproduced by mammalian cells (e.g., tissue-type plasminogen activatorsignal sequence, as described, for example, in U.S. Pat. No. 5,641,655)is useful for expression of Zcytor16 in recombinant mammalian hosts, ayeast signal sequence is preferred for expression in yeast cells.Examples of suitable yeast signal sequences are those derived from yeastmating phermone α-factor (encoded by the MFα1 gene), invertase (encodedby the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene). See,for example, Romanos et al, “Expression of Cloned Genes in Yeast,” inDNA Cloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(eds.), pages 123-167 (Oxford University Press 1995).

Zcytor16 monomeric, homodimeric, heterodimeric and multimeric receptorpolypeptides can be prepared by expressing a truncated DNA encoding theextracellular domain, for example, a polypeptide which contains SEQ IDNO:13, amino acids 22-210 of SEQ ID NO:2, or the corresponding region ofa non-human receptor. It is preferred that the extracellular domainpolypeptides be prepared in a form substantially free of transmembraneand intracellular polypeptide segments. To direct the export of thereceptor domain from the host cell, the receptor DNA is linked to asecond DNA segment encoding a secretory peptide, such as a t-PAsecretory peptide. To facilitate purification of the secreted receptordomain, a C-terminal extension, such as a poly-histidine tag, substanceP, Flag™ peptide (Hopp et al, Biotechnology 6:1204-1210, (1988);available from Eastman Kodak Co., New Haven, Conn.) or anotherpolypeptide or protein for which an antibody or other specific bindingagent is available, can be fused to the receptor polypeptide. Moreover,heterodimeric and multimeric non-zcytor16 subunit extracellular cytokinebinding domains are a also prepared as above.

In an alternative approach, a receptor extracellular domain of zcytor16or other class I or II cytokine receptor component can be expressed as afusion with immunoglobulin heavy chain constant regions, typically anF_(c) fragment, which contains two constant region domains and a hingeregion but lacks the variable region (See, Sledziewski, A Z et al., U.S.Pat. Nos. 6,018,026 and 5,750,375). The soluble zcytor16, solublezcytor16/CRF2-4 heterodimers, and monomeric, homodimeric, heterodimericand multimeric polypeptides of the present invention include suchfusions. Such fusions are typically secreted as multimeric moleculeswherein the Fc portions are disulfide bonded to each other and tworeceptor polypeptides are arrayed in closed proximity to each other.Fusions of this type can be used to affinity purify the cognate ligandfrom solution, as an in vitro assay tool, to block signals in vitro byspecifically titrating out ligand, and as antagonists in vivo byadministering them parenterally to bind circulating ligand and clear itfrom the circulation. To purify ligand, a Zcytor16-Ig chimera is addedto a sample containing the ligand (e.g., cell-conditioned culture mediaor tissue extracts) under conditions that facilitate receptor-ligandbinding (typically near-physiological temperature, pH, and ionicstrength). The chimera-ligand complex is then separated by the mixtureusing protein A, which is immobilized on a solid support (e.g.,insoluble resin beads). The ligand is then eluted using conventionalchemical techniques, such as with a salt or pH gradient. In thealternative, the chimera itself can be bound to a solid support, withbinding and elution carried out as above. The chimeras may be used invivo to regulate inflammatory responses including acute phase responsessuch as serum amyloid A (SAA), C-reactive protein (CRP), and the like.Chimeras with high binding affinity are administered parenterally (e.g.,by intramuscular, subcutaneous or intravenous injection). Circulatingmolecules bind ligand and are cleared from circulation by normalphysiological processes. For use in assays, the chimeras are bound to asupport via the F_(c) region and used in an ELISA format.

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a soluble zcytor16 receptor or solublezcytor16 heterodimeric polypeptide, such as soluble zcytor16/CRF2-4, canbe prepared as a fusion to a dimerizing protein as disclosed in U.S.Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains, e.g., IgG γ1, andthe human κ light chain. Immunoglobulin-soluble zcytor16 receptor orimmunoglobulin-soluble zcytor16 heterodimeric or multimeric polypeptide,such as immunoglobulin-soluble zcytor16/CRF2-4 fusions can be expressedin genetically engineered cells to produce a variety of multimericzcytor16 receptor analogs. Auxiliary domains can be fused to solublezcytor16 receptor or soluble zcytor16 heterodimeric or multimericpolypeptides, such as soluble zcytor16/CRF2-4 to target them to specificcells, tissues, or macromolecules (e.g., collagen, or cells expressingthe zcytor16 ligand, IL-TIF). A zcytor16 polypeptide can be fused to twoor more moieties, such as an affinity tag for purification and atargeting domain. Polypeptide fusions can also comprise one or morecleavage sites, particularly between domains. See, Tuan et al.,Connective Tissue Research 34:1-9, 1996.

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, Zcytor16can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a Zcytor16fusion protein comprising a maltose binding protein polypeptide can beisolated with an amylose resin column, while a fusion protein comprisingthe C-terminal end of a truncated Protein A gene can be purified usingIgG-SEPHAROSE®. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al., “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2^(rd) Edition,Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al, Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

The present invention also contemplates that the use of the secretorysignal sequence contained in the Zcytor16 polypeptides of the presentinvention to direct other polypeptides into the secretory pathway. Asignal fusion polypeptide can be made wherein a secretory signalsequence derived from amino acid residues 1 to 21, or 1 to 22, of SEQ IDNO:2 is operably linked to another polypeptide using methods known inthe art and disclosed herein. The secretory signal sequence contained inthe fusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein, such as a receptor. Suchfusions may be used in a transgenic animal or in a cultured recombinanthost to direct peptides through the secretory pathway. With regard tothe latter, exemplary polypeptides include pharmaceutically activemolecules such as Factor VIa, proinsulin, insulin, follicle stimulatinghormone, tissue type plasminogen activator, tumor necrosis factor,interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15), colonystimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF)and granulocyte macrophage-colony stimulating factor (GM-CSF)),interferons (e.g., interferons-α, -β, -γ, -ω, -δ, and -τ), the stem cellgrowth factor designated “S1 factor,” erythropoietin, andthrombopoietin. The Zcytor16 secretory signal sequence contained in thefusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Fusion proteins comprising aZcytor16 secretory signal sequence can be constructed using standardtechniques.

Another form of fusion protein comprises a Zcytor16 polypeptide and animmunoglobulin heavy chain constant region, typically an F_(c) fragment,which contains two or three constant region domains and a hinge regionbut lacks the variable region. As an illustration, Chang et al., U.S.Pat. No. 5,723,125, describe a fusion protein comprising a humaninterferon and a human immunoglobulin Fc fragment. The C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:4). In this fusion protein, an illustrative Fc moiety is ahuman γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a Zcytor16 fusion protein that comprises a Zcytor16 moietyand a human Fc fragment, wherein the C-terminus of the Zcytor16 moietyis attached to the N-terminus of the Fc fragment via a peptide linker,such as a peptide consisting of the amino acid sequence of SEQ ID NO:4.The Zcytor16 moiety can be a Zcytor16 molecule or a fragment thereof.For example, a fusion protein can comprise amino acid residues 22 to 210of SEQ ID NO:2 and an Fc fragment (e.g., a human Fc fragment).

In another variation, a Zcytor16 fusion protein comprises an IgGsequence, a Zcytor16 moiety covalently joined to the aminoterminal endof the IgG sequence, and a signal peptide that is covalently joined tothe aminoterminal of the Zcytor16 moiety, wherein the IgG sequenceconsists of the following elements in the following order: a hingeregion, a CH₂ domain, and a CH₃ domain. Accordingly, the IgG sequencelacks a CH₁ domain. The Zcytor16 moiety displays a Zcytor16 activity, asdescribed herein, such as the ability to bind with a Zcytor16 ligand.This general approach to producing fusion proteins that comprise bothantibody and nonantibody portions has been described by LaRochelle etal, EP 742830 (WO 95/21258).

Fusion proteins comprising a Zcytor16 moiety and an Fc moiety can beused, for example, as an in vitro assay tool. For example, the presenceof a Zcytor16 ligand in a biological sample can be detected using aZcytor16-immunoglobulin fusion protein, in which the Zcytor16 moiety isused to bind the ligand, and a macromolecule, such as Protein A oranti-Fc antibody, is used to bind the fusion protein to a solid support.Such systems can be used to identify agonists and antagonists thatinterfere with the binding of a Zcytor16 ligand to its receptor.

Other examples of antibody fusion proteins include polypeptides thatcomprise an antigen-binding domain and a Zcytor16 fragment that containsa Zcytor16 extracellular domain. Such molecules can be used to targetparticular tissues for the benefit of Zcytor16 binding activity.

The present invention further provides a variety of other polypeptidefusions. For example, part or all of a domain(s) conferring a biologicalfunction can be swapped between Zcytor16 of the present invention withthe functionally equivalent domain(s) from another member of thecytokine receptor family. Polypeptide fusions can be expressed inrecombinant host cells to produce a variety of Zcytor16 fusion analogs.A Zcytor16 polypeptide can be fused to two or more moieties or domains,such as an affinity tag for purification and a targeting domain.Polypeptide fusions can also comprise one or more cleavage sites,particularly between domains. See, for example, Tuan et al, ConnectiveTissue Research 34:1 (1996).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

Zcytor16 polypeptides can be used to identify and to isolate Zcytor16ligands. For example, proteins and peptides of the present invention canbe immobilized on a column and used to bind ligands from a biologicalsample that is run over the column (Hermanson et al (eds.), ImmobilizedAffinity Ligand Techniques, pages 195-202 (Academic Press 1992)). Assuch, zcytor16 polypeptides of the present invention can be used toidentify and isolate IL-TIF for either diagnostic, or productionpurposes.

The activity of a Zcytor16 polypeptide can also be observed by asilicon-based biosensor microphysiometer, which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the CYTOSENSOR Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method (see, for example, McConnell et al.,Science 257:1906 (1992), Pitchford et al., Meth. Enzymol. 228:84 (1997),Arimilli et al, J. Immunol. Meth. 212:49 (1998), Van Liefde et al, Eur.J. Pharmacol 346:87 (1998)). The microphysiometer can be used forassaying eukaryotic, prokaryotic, adherent, or non-adherent cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including agonists, ligands, or antagonists of Zcytor16.

For example, the microphysiometer is used to measure responses of anZcytor16-expressing eukaryotic cell, compared to a control eukaryoticcell that does not express Zcytor16 polypeptide. Suitable cellsresponsive to Zcytor16-modulating stimuli include recombinant host cellscomprising a Zcytor16 expression vector, and cells that naturallyexpress Zcytor16. Extracellular acidification provides one measure for aZcytor16-modulated cellular response. In addition, this approach can beused to identify ligands, agonists, and antagonists of Zcytor16 ligand,IL-TIF. For example, a molecule can be identified as an agonist ofZcytor16 ligand by providing cells that express a Zcytor16 polypeptide,culturing a first portion of the cells in the absence of the testcompound, culturing a second portion of the cells in the presence of thetest compound, and determining whether the second portion exhibits acellular response, in comparison with the first portion.

Alternatively, a solid phase system can be used to identify a Zcytor16ligand, or an agonist or antagonist of a Zcytor16 ligand. For example, aZcytor16 polypeptide or Zcytor16 fusion protein, or zcytor16 monomeric,homodimeric, heterodimeric or multimeric soluble receptor can beimmobilized onto the surface of a receptor chip of a commerciallyavailable biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden).The use of this instrument is disclosed, for example, by Karlsson,Immunol Methods 145:229 (1991), and Cunningham and Wells, J. Mol. Biol.234:554 (1993).

In brief, a Zcytor16 polypeptide or fusion protein is covalentlyattached, using amine or sulfhydryl chemistry, to dextran fibers thatare attached to gold film within a flow cell. A test sample is thenpassed through the cell. If a ligand is present in the sample, it willbind to the immobilized polypeptide or fusion protein, causing a changein the refractive index of the medium, which is detected as a change insurface plasmon resonance of the gold film. This system allows thedetermination of on- and off-rates, from which binding affinity can becalculated, and assessment of stoichiometry of binding. This system canalso be used to examine antibody-antigen interactions, and theinteractions of other complement/anti-complement pairs.

Zcytor16 binding domains can be further characterized by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids of Zcytor16 ligand agonists. See, for example, de Voset al, Science 255:306 (1992), Smith et al, J. Mol. Biol 224:899 (1992),and Wlodaver et al, FEBS Lett. 309:59 (1992).

The present invention also contemplates chemically modified Zcytor16compositions, in which a Zcytor16 polypeptide is linked with a polymer.Illustrative Zcytor16 polypeptides are soluble polypeptides that lack afunctional transmembrane domain, such as a polypeptide consisting ofamino acid residues 22 to 210 of SEQ ID NO:2. Typically, the polymer iswater soluble so that the Zcytor16 conjugate does not precipitate in anaqueous environment, such as a physiological environment. An example ofa suitable polymer is one that has been modified to have a singlereactive group, such as an active ester for acylation, or an aldehydefor alkylation, In this way, the degree of polymerization can becontrolled. An example of a reactive aldehyde is polyethylene glycolpropionaldehyde, or mono-(C1-C10) alkoxy, or aryloxy derivatives thereof(see, for example, Harris, et al, U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Moreover, a mixture of polymers can beused to produce Zcytor16 conjugates.

Zcytor16 conjugates used for therapy can comprise pharmaceuticallyacceptable water-soluble polymer moieties. Suitable water-solublepolymers include polyethylene glycol (PEG), monomethoxy-PEG,mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG,tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonatePEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, dextran, cellulose, or other carbohydrate-based polymers.Suitable PEG may have a molecular weight from about 600 to about 60,000,including, for example, 5,000, 12,000, 20,000 and 25,000. A Zcytor16conjugate can also comprise a mixture of such water-soluble polymers.

One example of a Zcytor16 conjugate comprises a Zcytor16 moiety and apolyalkyl oxide moiety attached to the N-terminus of the Zcytor16moiety. PEG is one suitable polyalkyl oxide. As an illustration,Zcytor16 can be modified with PEG, a process known as “PEGylation.”PEGylation of Zcytor16 can be carried out by any of the PEGylationreactions known in the art (see, for example, EP 0 154 316, Delgado etal, Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992),Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis etal, Int J Hematol 68:1 (1998)). For example, PEGylation can be performedby an acylation reaction or by an alkylation reaction with a reactivepolyethylene glycol molecule. In an alternative approach, Zcytor16conjugates are formed by condensing activated PEG, in which a terminalhydroxy or amino group of PEG has been replaced by an activated linker(see, for example, Karasiewicz et al, U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a Zcytor16 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, the term “acylation” includes the following types of linkagesbetween Zcytor16 and a water soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated Zcytor16 byacylation will typically comprise the steps of (a) reacting a Zcytor16polypeptide with PEG (such as a reactive ester of an aldehyde derivativeof PEG) under conditions whereby one or more PEG groups attach toZcytor16, and (b) obtaining the reaction product(s). Generally, theoptimal reaction conditions for acylation reactions will be determinedbased upon known parameters and desired results. For example, the largerthe ratio of PEG:Zcytor16, the greater the percentage of polyPEGylatedZcytor16 product.

The product of PEGylation by acylation is typically a polyPEGylatedZcytor16 product, wherein the lysine ε-amino groups are PEGylated via anacyl linking group. An example of a connecting linkage is an amide.Typically, the resulting Zcytor16 will be at least 95% mono-, di-, ortri-pegylated, although some species with higher degrees of PEGylationmay be formed depending upon the reaction conditions. PEGylated speciescan be separated from unconjugated Zcytor16 polypeptides using standardpurification methods, such as dialysis, ultrafiltration, ion exchangechromatography, affinity chromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with Zcytor16 in the presence of a reducing agent. PEGgroups can be attached to the polypeptide via a —CH₂—NH group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of Zcytor16monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer Zcytor16 conjugate molecule can comprise the steps of: (a)reacting a Zcytor16 polypeptide with a reactive PEG under reductivealkylation conditions at a pH suitable to permit selective modificationof the α-amino group at the amino terminus of the Zcytor16, and (b)obtaining the reaction product(s). The reducing agent used for reductivealkylation should be stable in aqueous solution and able to reduce onlythe Schiff base formed in the initial process of reductive alkylation.Illustrative reducing agents include sodium borohydride, sodiumcyanoborohydride, dimethylamine borane, trimethylamine borane, andpyridine borane.

For a substantially homogenous population of monopolymer Zcytor16conjugates, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of Zcytor16. Such reaction conditions generally providefor pKa differences between the lysine amino groups and the α-aminogroup at the N-terminus. The pH also affects the ratio of polymer toprotein to be used. In general, if the pH is lower, a larger excess ofpolymer to protein will be desired because the less reactive theN-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer:Zcytor16 need not be aslarge because more reactive groups are available. Typically, the pH willfall within the range of 3 to 9, or 3 to 6. This method can be employedfor making zcytor16-comprising homodimeric, heterodimeric or multimericsoluble receptor conjugates.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to Zcytor16 will generallybe in the range of 1:1 to 100:1. Typically, the molar ratio ofwater-soluble polymer to Zcytor16 will be 1:1 to 20:1 forpolyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al, U.S. Pat. No. 5,382,657, Greenwald et al, U.S. Pat.No. 5,738,846, Nieforth et al, Clin. Pharmacol Ther. 59:636 (1996),Monkarsh et al, Anal Biochem. 247:434 (1997)). This method can beemployed for making zcytor16-comprising homodimeric, heterodimeric ormultimeric soluble receptor conjugates.

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

8. Isolation of Zcytor16 Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of Zcytor16 purified from natural sources (e.g.,tonsil tissue), synthetic Zcytor16 polypeptides, and recombinantZcytor16 polypeptides and fusion Zcytor16 polypeptides purified fromrecombinant host cells. In general, ammonium sulfate precipitation andacid or chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are suitable. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-SEPHAROSE® FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-SEPHAROSE® (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in Zcytor16 isolation and purification can bedevised by those of skill in the art. For example, anti-Zcytor16antibodies, obtained as described below, can be used to isolate largequantities of protein by immunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification. Moreover, theligand-binding properties of zcytor16 extracellular domain can beexploited for purification, for example, of zcytor16-comprising solublereceptors; for example, by using affinity chromatography wherein IL-TIFligand is bound to a column and the zcytor16-comprising receptor isbound and subsequently eluted using standard chromatography methods.

Zcytor16 polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. Zcytor16 polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; PEGylated ornon-PEGylated; and may or may not include an initial methionine aminoacid residue.

9. Production of Antibodies to Zcytor16 Proteins

Antibodies to Zcytor16 can be obtained, for example, using the productof a Zcytor16 expression vector or Zcytor16 isolated from a naturalsource as an antigen. Particularly useful anti-Zcytor16 antibodies “bindspecifically” with Zcytor16. Antibodies are considered to bespecifically binding if the antibodies exhibit at least one of thefollowing two properties: (1) antibodies bind to Zcytor16 with athreshold level of binding activity, and (2) antibodies do notsignificantly cross-react with polypeptides related to Zcytor16.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a Zcytor16 polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect Zcytor16, but notpresently known polypeptides using a standard Western blot analysis.Examples of known related polypeptides include known cytokine receptors.

Anti-Zcytor16 antibodies can be produced using antigenic Zcytor16epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2 or another amino acid sequence disclosed herein. However,peptides or polypeptides comprising a larger portion of an amino acidsequence of the invention, containing from 30 to 50 amino acids, or anylength up to and including the entire amino acid sequence of apolypeptide of the invention, also are useful for inducing antibodiesthat bind with Zcytor16. It is desirable that the amino acid sequence ofthe epitope-bearing peptide is selected to provide substantialsolubility in aqueous solvents (i.e., the sequence includes relativelyhydrophilic residues, while hydrophobic residues are typically avoided).Moreover, amino acid sequences containing proline residues may be alsobe desirable for antibody production.

As an illustration, potential antigenic sites in Zcytor16 wereidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al, J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Garnier et al, J. Mol. Biol120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Garnier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:2 would provide suitable antigenic peptides:amino acids 24 to 42 (“antigenic peptide 1”), amino acids 24 to 33(“antigenic peptide 2”), 37 to 42 (“antigenic peptide 3”), amino acids48 to 55 (“antigenic peptide 4”), amino acids 68 to 81 (“antigenicpeptide 5”), amino acids 88 to 97 (“antigenic peptide 6”), amino acids126 to 132 (“antigenic peptide 7”), amino acids 156 to 165 (“antigenicpeptide 8”), amino acids 178 to 185 (“antigenic peptide 9”), and aminoacids 216 to 227 (“antigenic peptide 10”). The present inventioncontemplates the use of any one of antigenic peptides 1 to 10 togenerate antibodies to Zcytor16. The present invention also contemplatespolypeptides comprising at least one of antigenic peptides 1 to 10.

Moreover, suitable antigens also include the zcytor16 polypeptidescomprising a zcytor16 cytokine binding, or extracellular domaindisclosed above in combination with another class I or II cytokineextracellular domain, such as those that form soluble zcytor16heterodimeric or multimeric polypeptides, such as solublezcytor16/CRF2-4, zcytor16/zcytor11, zcytor16/zcytor7, and the like.

Polyclonal antibodies to recombinant Zcytor16 protein or to Zcytor16isolated from natural sources can be prepared using methods well-knownto those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a Zcytor16 polypeptide canbe increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of Zcytor16 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like,” such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-Zcytor16 antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al, Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-Zcytor16 antibodies can be generated.Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art (see, for example, Kohler etal, Nature 256:495 (1975), Coligan et al (eds.), Current Protocols inImmunology, Vol 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al, “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a Zcytor16 gene product, verifying the presenceof antibody production by removing a serum sample, removing the spleento obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-Zcytor16 antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al, NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A SEPHAROSE®,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-Zcytor16 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al, Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal, Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al, Methods: A Companionto Methods in Enzymology 2:97 (1991) (also see, Bird et al, Science242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et al.,Bio/Technology 11:1271 (1993), and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toZcytor16 polypeptide in vitro, and selecting antibody display librariesin phage or similar vectors (for instance, through use of immobilized orlabeled Zcytor16 protein or peptide). Genes encoding polypeptides havingpotential Zcytor16 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al, U.S. Pat.No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kayet al., Phage Display of Peptides and Proteins (Academic Press, Inc.1996)) and random peptide display libraries and kits for screening suchlibraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the Zcytor16 sequences disclosed herein toidentify proteins which bind to Zcytor16.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zcytor16 antibody may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Carter et al, Proc. Nat'l Acad. Sci. USA 89:4285(1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J.Immun. 150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols(Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,”in Protein Engineering: Principles and Practice, Cleland et al (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zcytor16 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al, “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-Zcytor16 antibodies or antibodyfragments as immunogens with the techniques, described above. As anotheralternative, humanized anti-idiotype antibodies or subhuman primateanti-idiotype antibodies can be prepared using the above-describedtechniques. Methods for producing anti-idiotype antibodies aredescribed, for example, by Irie, U.S. Pat. No. 5,208,146, Greene, et.al, U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.77:1875 (1996).

10. Use of Zcytor16 Nucleotide Sequences to Detect Gene Expression andGene Structure

Nucleic acid molecules can be used to detect the expression of aZcytor16 gene in a biological sample. Suitable probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1, or a portion thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, or a portion thereof. Probemolecules may be DNA, RNA, oligonucleotides, and the like. As usedherein, the term “portion” refers to at least eight nucleotides to atleast 20 or more nucleotides. Illustrative probes bind with regions ofthe Zcytor16 gene that have a low sequence similarity to comparableregions in other cytokine receptor genes.

In a basic assay, a single-stranded probe molecule is incubated withRNA, isolated from a biological sample, under conditions of temperatureand ionic strength that promote base pairing between the probe andtarget Zcytor16 RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

In addition, as zcytor16 is spleen-specific, polynucleotide probes,anti-zcytor16 antibodies, and detection the presence of zcytor16polypeptides in tissue can be used to assess whether spleen tissue ispresent, for example, after surgery involving the excision of a diseasedor cancerous spleen. As such, the polynucleotides, polypeptides, andantibodies of the present invention can be used as an aid to determinewhether all spleen tissue is excised after surgery, for example, aftersurgery for spleen cancer. In such instances, it is especially importantto remove all potentially diseased tissue to maximize recovery from thecancer, and to minimize recurrence. Preferred embodiments includefluorescent, radiolabeled, or calorimetrically labeled antibodies, thatcan be used in situ.

Moreover, anti-zcytor16 antibodies and binding fragments can be used fortagging and sorting cells that specifically-express Zcytor16, such asmononuclear cells, lymphoid cells, e.g, activated CD4+ T-cells and CD19+B-cells, and other described herein. Such methods of cell tagging andsorting are well known in the art (see, e.g., “Molecular Biology of theCell”, 3^(rd) Ed., Albert, B. et al (Garland Publishing, London & NewYork, 1994). One of skill in the art would recognize the importance ofseparating cell tissue types to study cells, and the use of antibodiesto separate specific cell tissue types. Basically, antibodies that bindto the surface of a cell type are coupled to various matrices such ascollagen, polysaccharide beads, or plastic to form an affinity surfaceto which only cells recognized by the antibodies will adhere. The boundcells are then recovered by conventional techniques. Other methodsinvolve separating cells by a fluorescence-activated cell sorter (FACS).In this technique one labels cells with antibodies that are coupled to afluorescent dye. The labeled cells are then separated from unlabeledcells in a FACS machine. In FACS sorting individual cells traveling insingle file pass through a laser beam and the fluorescence of each cellis measured. Slightly further down-stream, tiny droplets, mostcontaining either one or no cells, are formed by a vibrating nozzle. Thedroplets containing a single cell are automatically give a positive ornegative charge at the moment of formation, depending on whether thecell they contain is fluorescent, and then deflected by a strongelectric field into an appropriate container. Such machines can select 1cell in 1000 and sort about 5000 cells each second. This produces auniform population of cells for cell culture.

One of skill in the art would recognize that the antibodies to theZcytor16 polypeptides of the present invention are useful, because notall tissue types express the Zcytor16 receptor and because it isimportant that biologists be able to separate specific cell types forfurther study and/or therapeutic re-implantation into the body. This isparticularly relevant in cells such as immune cells, wherein zcytor16 isexpressed.

Moreover, use of Zcytor16 polynucleotide probes, anti-zcytor16antibodies, and detection the presence of zcytor16 polypeptides intissue can be used in the diagnosis and/or prevention of spontaneousabortions, or to monitor placental health and function. Since Zcytor16is expressed in the placenta, it could play a role in the criticalfunctions of placenta, such as proliferation or survival of trophoblastcells, and the like. Thus, zcytor16 could be essential for the functionof the placenta, thus maturation of embryos. Therefore, a supplement ofZcytor16 polypeptide, or anti-zcytor16 antibodies may be beneficial inthe prevention and treatment of certain types of spontaneous abortions,or premature birth of babies caused by abnormal expression of Zcytor16in the placenta, or as a diagnostic to assess the function of theplacenta. For example, as zcytor16 is normally expressed in placenta,the absence of zcytor16 expression may be indicative of abnormalplacenta function.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵S. Alternatively, Zcytor16RNA can be detected with a nonradioactive hybridization method (see, forexample, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Zcytor16 oligonucleotide probes are also useful for in vivo diagnosis.As an illustration, ¹⁸F-labeled oligonucleotides can be administered toa subject and visualized by positron emission tomography (Tavitian etal, Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

PCR primers can be designed to amplify a portion of the Zcytor16 genethat has a low sequence similarity to a comparable region in otherproteins, such as other cytokine receptor proteins.

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated withZcytor16 primers (see, for example, Wu et al. (eds.), “Rapid Isolationof Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology,pages 15-28 (CRC Press, Inc. 1997)). PCR is then performed and theproducts are analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the gunadinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or Zcytor16 anti-sense oligomers. Oligo-dT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Zcytor16 sequences are amplified bythe polymerase chain reaction using two flanking oligonucleotide primersthat are typically 20 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zcytor16 probe, andexamined by autoradiography. Additional alternative approaches includethe use of digoxigenin-labeled deoxyribonucleic acid triphosphates toprovide chemiluminescence detection, and the C-TRAK calorimetric assay.

Another approach for detection of Zcytor16 expression is cycling probetechnology (CPT), in which a single-stranded DNA target binds with anexcess of DNA-RNA-DNA chimeric probe to form a complex, the RNA portionis cleaved with RNAase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al, J. Clin. MicrobioAl 34:2985(1996), Bekkaoui et al., Biotechniques 20:240 (1996)). Alternativemethods for detection of Zcytor16 sequences can utilize approaches suchas nucleic acid sequence-based amplification (NASBA), cooperativeamplification of templates by cross-hybridization (CATCH), and theligase chain reaction (LCR) (see, for example, Marshall et al., U.S.Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161 (1996),Ehricht et al, Eur. J. Biochem. 243:358 (1997), and Chadwick et al., J.Virol Methods 70:59 (1998)). Other standard methods are known to thoseof skill in the art.

Zcytor16 probes and primers can also be used to detect and to localizeZcytor16 gene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)). Suitable test samples include blood, urine,saliva, tissue biopsy, and autopsy material.

The Zcytor16 gene resides in human chromosome 6q24.1-25.2. This regionis associated with various disorders, including insulin dependentdiabetes mellitus, retinal cone dystrophy, breast cancer, and Parkinsondisease. Moreover, defects in the zcytor16 locus itself may result in aheritable human disease states as discussed herein. One of skill in theart would appreciate that defects in cytokine receptors are known tocause particular diseases in humans. For example, polymorphisms ofcytokine receptors are associated with pulmonary alveolar proteinosis,familial periodic fever, and erythroleukemia. Moreover, growth hormonereceptor mutation results in dwarfism (Amselem, S et al., New Eng. J.Med. 321: 989-995, 1989), IL-2 receptor gamma mutation results in severecombined immunodeficiency (SCID) (Noguchi, M et al., Cell 73: 147-157,1993), c-Mpl mutation results in thrombocytopenia (Ihara, K et al.,Proc. Nat. Acad. Sci. 96: 3132-3136, 1999), and severe mycobacterial andSalmonella infections result in interleukin-12 receptor-deficientpatients (de Jong, R et al., Science 280: 1435-1438, 1998), amongstothers. Thus, similarly, defects in zcytor16 can cause a disease stateor susceptibility to disease or infection. As the Zcytor16 gene islocated at 6q24.1-25.2, zcytor16 polynucleotide probes can be used todetect chromosome 6q24.1-25.2 loss, trisomy, duplication ortranslocation associated with human diseases, such as inflammatorydiseases, chronic inflammation, dysfunction of inflammatory response,immune cell cancers, bone marrow cancers, spleen cancers, prostatecancer, thyroid, parathyroid or other cancers, or immune diseases.Moreover, molecules of the present invention, such as the polypeptides,antagonists, agonists, polynucleotides and antibodies of the presentinvention would aid in the detection, diagnosis prevention, andtreatment associated with a zcytor16 genetic defect. Thus, Zcytor16nucleotide sequences can be used in linkage-based testing for variousdiseases, and to determine whether a subject's chromosomes contain amutation in the Zcytor16 gene. Detectable chromosomal aberrations at theZcytor16 gene locus include, but are not limited to, aneuploidy, genecopy number changes, loss of heterogeneity of 6q24.1-25.2, translocationin 6q24.1-25.2, insertions, deletions, restriction site changes andrearrangements. Of particular interest are genetic alterations thatinactivate a Zcytor16 gene, or gross chromosomal alterations in andaround the zcytor16 locus.

Similarly, defects in the Zcytor16 gene itself may result in a heritablehuman disease state. Moreover, one of skill in the art would appreciatethat defects in cytokine receptors are known to cause disease states inhumans. For example, growth hormone receptor mutation results indwarfism (Amselem, S et al., New Eng. J. Med. 321: 989-995, 1989), IL-2receptor gamma mutation results in severe combined immunodeficiency(SCID) (Noguchi, M et al., Cell 73: 147-157, 1993), c-Mpl mutationresults in thrombocytopenia (Ihara, K et al., Proc. Nat. Acad. Sci. 96:3132-3136, 1999), and severe mycobacterial and Salmonella infectionsresult in interleukin-12 receptor-deficient patients (de Jong, R et al.,Science 280: 1435-1438, 1998), amongst others. Thus, similarly, defectsin zcytor16 can cause a disease state or susceptibility to disease orinfection. As, zcytor16 is a cytokine receptor within a chromosomalregion where aberrations may be involved in cancer, and is shown to beexpressed in ovarian cancer, the molecules of the present inventioncould also be directly involved in cancer formation or metastasis. Asthe Zcytor16 gene is located at the 6q24.1-25.2 region zcytor16,polynucleotide probes can be used to detect chromosome 6q24.1-25.2 loss,loss of heterogeneity (LOH), trisomy, duplication or translocationassociated with human diseases, such as immune cell cancers,neuroblastoma, bone marrow cancers, thyroid, parathyroid, prostate,melanoma, or other cancers, or immune diseases. Moreover, molecules ofthe present invention, such as the polypeptides, antagonists, agonists,polynucleotides and antibodies of the present invention would aid in thedetection, diagnosis prevention, and treatment associated with aZcytor16 genetic defect.

A diagnostic could assist physicians in determining the type of diseaseand appropriate associated therapy, or assistance in genetic counseling.As such, the inventive anti-zcytor16 antibodies, polynucleotides, andpolypeptides can be used for the detection of zcytor16 polypeptide, mRNAor anti-zcytor16 antibodies, thus serving as markers and be directlyused for detecting or genetic diseases or cancers, as described herein,using methods known in the art and described herein. Further, zcytor16polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 6q24.1-25.2 deletions and translocationsassociated with human diseases, other translocations involved withmalignant progression of tumors or other 6q24.1-25.2 mutations, whichare expected to be involved in chromosome rearrangements in malignancy;or in other cancers, or in spontaneous abortion. Similarly, zcytor16polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 6q24.1-25.2 trisomy and chromosome lossassociated with human diseases. Thus, zcytor16 polynucleotide probes canbe used to detect abnormalities or genotypes associated with thesedefects.

As discussed above, defects in the Zcytor16 gene itself may result in aheritable human disease state. For example, zcytor16 expression iselevated in several tissue-specific human cancers, as described herein.Molecules of the present invention, such as the polypeptides,antagonists, agonists, polynucleotides and antibodies of the presentinvention would aid in the detection, diagnosis prevention, andtreatment associated with a zcytor16 genetic defect. In addition,zcytor16 polynucleotide probes can be used to detect allelic differencesbetween diseased or non-diseased individuals at the zcytor16 chromosomallocus. As such, the zcytor16 sequences can be used as diagnostics inforensic DNA profiling.

In general, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Analytical probes will be generally at least 20 nt in length,although somewhat shorter probes can be used (e.g., 14-17 nt). PCRprimers are at least 5 nt in length, preferably 15 or more, morepreferably 20-30 nt. For gross analysis of genes, or chromosomal DNA, azcytor16 polynucleotide probe may comprise an entire exon or more. Exonsare readily determined by one of skill in the art by comparing zcytor16sequences (SEQ ID NO:1) with the human genomic DNA for zcytor16. Ingeneral, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Most diagnostic methods comprise the steps of (a) obtaining agenetic sample from a potentially diseased patient, diseased patient orpotential non-diseased carrier of a recessive disease allele; (b)producing a first reaction product by incubating the genetic sample witha zcytor16 polynucleotide probe wherein the polynucleotide willhybridize to complementary polynucleotide sequence, such as in RFLPanalysis or by incubating the genetic sample with sense and antisenseprimers in a PCR reaction under appropriate PCR reaction conditions;(iii) Visualizing the first reaction product by gel electrophoresisand/or other known method such as visualizing the first reaction productwith a zcytor16 polynucleotide probe wherein the polynucleotide willhybridize to the complementary polynucleotide sequence of the firstreaction; and (iv) comparing the visualized first reaction product to asecond control reaction product of a genetic sample from wild typepatient. A difference between the first reaction product and the controlreaction product is indicative of a genetic abnormality in the diseasedor potentially diseased patient, or the presence of a heterozygousrecessive carrier phenotype for a non-diseased patient, or the presenceof a genetic defect in a tumor from a diseased patient, or the presenceof a genetic abnormality in a fetus or pre-implantation embryo. Forexample, a difference in restriction fragment pattern, length of PCRproducts, length of repetitive sequences at the zcytor16 genetic locus,and the like, are indicative of a genetic abnormality, geneticaberration, or allelic difference in comparison to the normal wild typecontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated form any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO:1, thecomplement of SEQ ID NO:1, or an RNA equivalent thereof. Such methods ofshowing genetic linkage analysis to human disease phenotypes are wellknown in the art. For reference to PCR based methods in diagnostics seesee, generally, Mathew (ed.), Protocols in Human Molecular Genetics(Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methodsand Applications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

Aberrations associated with the Zcytor16 locus can be detected usingnucleic acid molecules of the present invention by employing moleculargenetic techniques, such as restriction fragment length polymorphism(RFLP) analysis, short tandem repeat (STR) analysis employing PCRtechniques, amplification-refractory mutation system analysis (ARMS),single-strand conformation polymorphism (SSCP) detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis (FAMA), and other genetic analysis techniques known inthe art (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)).

The protein truncation test is also useful for detecting theinactivation of a gene in which translation-terminating mutationsproduce only portions of the encoded protein (see, for example,Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to thisapproach, RNA is isolated from a biological sample, and used tosynthesize cDNA. PCR is then used to amplify the Zcytor16 targetsequence and to introduce an RNA polymerase promoter, a translationinitiation sequence, and an in-frame ATG triplet. PCR products aretranscribed using an RNA polymerase, and the transcripts are translatedin vitro with a T7-coupled reticulocyte lysate system. The translationproducts are then fractionated by SDS-PAGE to determine the lengths ofthe translation products. The protein truncation test is described, forexample, by Dracopoli et al. (eds.), Current Protocols in HumanGenetics, pages 9.11.1-9.11.18 (John Wiley & Sons 1998).

The present invention also contemplates kits for performing a diagnosticassay for Zcytor16 gene expression or to detect mutations in theZcytor16 gene. Such kits comprise nucleic acid probes, such asdouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1, or a portion thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, or a portion thereof. Probemolecules may be DNA, RNA, oligonucleotides, and the like. Kits maycomprise nucleic acid primers for performing PCR.

Such kits can contain all the necessary elements to perform a nucleicacid diagnostic assay described above. A kit will comprise at least onecontainer comprising a Zcytor16 probe or primer. The kit may alsocomprise a second container comprising one or more reagents capable ofindicating the presence of Zcytor16 sequences. Examples of suchindicator reagents include detectable labels such as radioactive labels,fluorochromes, chemiluminescent agents, and the like. A kit may alsocomprise a means for conveying to the user that the Zcytor16 probes andprimers are used to detect Zcytor16 gene expression. For example,written instructions may state that the enclosed nucleic acid moleculescan be used to detect either a nucleic acid molecule that encodesZcytor16, or a nucleic acid molecule having a nucleotide sequence thatis complementary to a Zcytor16-encoding nucleotide sequence. The writtenmaterial can be applied directly to a container, or the written materialcan be provided in the form of a packaging insert.

11. Use of Anti-Zcytor16 Antibodies to Detect Zcytor16 or AntagonizeZcytor16 Binding to IL-TIF

The present invention contemplates the use of anti-Zcytor16 antibodiesto screen biological samples in vitro for the presence of Zcytor16. Inone type of in vitro assay, anti-Zcytor16 antibodies are used in liquidphase. For example, the presence of Zcytor16 in a biological sample canbe tested by mixing the biological sample with a trace amount of labeledZcytor16 and an anti-Zcytor16 antibody under conditions that promotebinding between Zcytor16 and its antibody. Complexes of Zcytor16 andanti-Zcytor16 in the sample can be separated from the reaction mixtureby contacting the complex with an immobilized protein which binds withthe antibody, such as an Fc antibody or Staphylococcus protein A. Theconcentration of Zcytor16 in the biological sample will be inverselyproportional to the amount of labeled Zcytor16 bound to the antibody anddirectly related to the amount of free labeled Zcytor16. Illustrativebiological samples include blood, urine, saliva, tissue biopsy, andautopsy material.

Alternatively, in vitro assays can be performed in which anti-Zcytor16antibody is bound to a solid-phase carrier. For example, antibody can beattached to a polymer, such as aminodextran, in order to link theantibody to an insoluble support such as a polymer-coated bead, a plateor a tube. Other suitable in vitro assays will be readily apparent tothose of skill in the art.

In another approach, anti-Zcytor16 antibodies can be used to detectZcytor16 in tissue sections prepared from a biopsy specimen. Suchimmunochemical detection can be used to determine the relative abundanceof Zcytor16 and to determine the distribution of Zcytor16 in theexamined tissue. General immunochemistry techniques are well established(see, for example, Ponder, “Cell Marking Techniques and TheirApplication,” in Mammalian Development: A Practical Approach, Monk(ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8,Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), andManson (ed.), Methods In Molecular Biology, Vol 10: ImmunochemicalProtocols (The Humana Press, Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-Zcytor16 antibody, and then contacting thebiological sample with a detectably labeled molecule which binds to theantibody. For example, the detectably labeled molecule can comprise anantibody moiety that binds to anti-Zcytor16 antibody. Alternatively, theanti-Zcytor16 antibody can be conjugated with avidin/streptavidin (orbiotin) and the detectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

Alternatively, an anti-Zcytor16 antibody can be conjugated with adetectable label to form an anti-Zcytor16 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵, ¹¹³, ³⁵S and ¹⁴C.

Anti-Zcytor16 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-Zcytor16 immunoconjugates can be detectably labeledby coupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemi-luminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-Zcytor16immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-Zcytor16 immunoconjugates can be detectably labeledby linking an anti-Zcytor16 antibody component to an enzyme. When theanti-Zcytor16-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-Zcytor16 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al, Clin. Chim. Acta 70:1 (1976), Schurs etal, Clin. Chim. Acta 81:1 (1977), Shih et al, Int'l J. Cancer 46:1101(1990), Stein et al, Cancer Res. 50:1330 (1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-Zcytor16 antibodies that have beenconjugated with avidin, streptavidin, and biotin (see, for example,Wilchek et al (eds.), “Avidin-Biotin Technology,” Methods In Enzymology,Vol 184 (Academic Press 1990), and Bayer et al, “ImmunochemicalApplications of Avidin-Biotin Technology,” in Methods In MolecularBiology, Vol 10, Manson (ed.), pages 149-162 (The Humana Press, Inc.1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

The present invention also contemplates kits for performing animmunological diagnostic assay for Zcytor16 gene expression. Such kitscomprise at least one container comprising an anti-Zcytor16 antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of Zcytor16antibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that Zcytor16 antibodies or antibody fragments areused to detect Zcytor16 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect Zcytor16. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to soluble zcytor16monomeric receptor or soluble zcytor16 homodimeric, heterodimeric ormultimeric polypeptides, and selection of antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled soluble zcytor16 monomeric receptor or soluble zcytor16homodimeric, heterodimeric or multimeric polypeptides). Genes encodingpolypeptides having potential binding domains such as soluble zcytor16monomeric receptor or soluble zcytor16 homodimeric, heterodimeric ormultimeric polypeptide can be obtained by screening random peptidelibraries displayed on phage (phage display) or on bacteria, such as E.coli. Nucleotide sequences encoding the polypeptides can be obtained ina number of ways, such as through random mutagenesis and randompolynucleotide synthesis. These random peptide display libraries can beused to screen for peptides which interact with a known target which canbe a protein or polypeptide, such as a ligand or receptor, a biologicalor synthetic macromolecule, or organic or inorganic substances.Techniques for creating and screening such random peptide displaylibraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409;Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No.5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) and random peptidedisplay libraries and kits for screening such libraries are availablecommercially, for instance from Clontech (Palo Alto, Calif.), InvitrogenInc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) andPharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptidedisplay libraries can be screened using the soluble zcytor16 monomericreceptor or soluble zcytor16 homodimeric, heterodimeric or multimericpolypeptide sequences disclosed herein to identify proteins which bindto zcytor16-comprising receptor polypeptides. These “bindingpolypeptides,” which interact with soluble zcytor16-comprising receptorpolypeptides, can be used for tagging cells; for isolating homologpolypeptides by affinity purification; they can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like.These binding polypeptides can also be used in analytical methods suchas for screening expression libraries and neutralizing activity, e.g.,for blocking interaction between IL-TIF ligand and receptor, or viralbinding to a receptor. The binding polypeptides can also be used fordiagnostic assays for determining circulating levels of solublezcytor16-comprising receptor polypeptides; for detecting or quantitatingsoluble or non-soluble zcytor16-comprising receptors as marker ofunderlying pathology or disease. These binding polypeptides can also actas “antagonists” to block soluble or membrane-bound zcytor16 monomericreceptor or zcytor16 homodimeric, heterodimeric or multimericpolypeptide binding (e.g. to ligand) and signal transduction in vitroand in vivo. Again, these binding polypeptides serve as anti-zcytor16monomeric receptor or anti-zcytor16 homodimeric, heterodimeric ormultimeric polypeptides and are useful for inhibiting IL-TIF activity,as well as receptor activity or protein-binding. Antibodies raised tothe natural receptor complexes of the present invention may be preferredembodiments, as they may act more specifically against the IL-TIF, ormore potently than antibodies raised to only one subunit. Moreover, theantagonistic and binding activity of the antibodies of the presentinvention can be assayed in the IL-TIF proliferation, signal trap,luciferase or binding assays in the presence of IL-TIF andzcytor16-comprising soluble receptors, and other biological orbiochemical assays described herein.

Antibodies to monomeric zcytor16 receptor or zcytor16 homodimeric,heterodimeric or multimeric zcytor16-containing receptors may be usedfor tagging cells that express zcytor16 receptors; for isolating solublezcytor16-comprising receptor polypeptides by affinity purification; fordiagnostic assays for determining circulating levels of solublezcytor16-comprising receptor polypeptides; for detecting or quantitatingsoluble zcytor16-comprising receptors as marker of underlying pathologyor disease; in analytical methods employing FACS; for screeningexpression libraries; for generating anti-idiotypic antibodies that canact as IL-TIF agonists; and as neutralizing antibodies or as antagoniststo block zcytor16 receptor function, or to block IL-TIF activity invitro and in vivo. Suitable direct tags or labels include radionuclides,enzymes, substrates, cofactors, biotin, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like; indirect tagsor labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Antibodies herein mayalso be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to solublezcytor16-comprising receptor polypeptides, or fragments thereof may beused in vitro to detect denatured or non-denatured zcytor16-comprisingreceptor polypeptides or fragments thereof in assays, for example,Western Blots or other assays known in the art.

Antibodies to soluble zcytor16 receptor or soluble zcytor16 homodimeric,heterodimeric or multimeric receptor polypeptides are useful for taggingcells that express the corresponding receptors and assaying theirexpression levels, for affinity purification, within diagnostic assaysfor determining circulating levels of receptor polypeptides, analyticalmethods employing fluorescence-activated cell sorting. Moreover,divalent antibodies, and anti-idiotypic antibodies may be used asagonists to mimic the effect of the zcytor16 ligand, IL-TIF.

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize soluble zcytor16 receptor orsoluble zcytor16 homodimeric, heterodimeric or multimeric receptorpolypeptides of the present invention can be used to identify or treattissues or organs that express a corresponding anti-complementarymolecule (i.e., a zcytor16-comprising soluble or membrane-boundreceptor). More specifically, antibodies to soluble zcytor16-comprisingreceptor polypeptides, or bioactive fragments or portions thereof, canbe coupled to detectable or cytotoxic molecules and delivered to amammal having cells, tissues or organs that express thezcytor16-comprising receptor such as zcytor16-expressing cancers, orcertain disease states.

Suitable detectable molecules may be directly or indirectly attached topolypeptides that bind zcytor16-comprising receptor polypeptides, suchas “binding polypeptides,” (including binding peptides disclosed above),antibodies, or bioactive fragments or portions thereof. Suitabledetectable molecules include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent markers, chemiluminescent markers,magnetic particles and the like. Suitable cytotoxic molecules may bedirectly or indirectly attached to the polypeptide or antibody, andinclude bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Binding polypeptidesor antibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the binding polypeptide or antibody portion. For thesepurposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the binding polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the fusion protein including only a singledomain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, zcytor16 binding polypeptide-cytokine orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, spleen, pancreatic, blood,lymphoid, colon, and bone marrow cancers), if the bindingpolypeptide-cytokine or anti-zcytor16 receptor antibody targets thehyperproliferative cell (See, generally, Hornick et al., Blood89:4437-47, 1997). The described fusion proteins enable targeting of acytokine to a desired site of action, thereby providing an elevatedlocal concentration of cytokine. Suitable anti-zcytor16 monomer,homodimer, heterodimer or multimer antibodies target an undesirable cellor tissue (i.e., a tumor or a leukemia), and the fused cytokine mediatesimproved target cell lysis by effector cells. Suitable cytokines forthis purpose include interleukin 2 and granulocyte-macrophagecolony-stimulating factor (GM-CSF), for instance.

Alternatively, zcytor16 receptor binding polypeptides or antibody fusionproteins described herein can be used for enhancing in vivo killing oftarget tissues by directly stimulating a zcytor16 receptor-modulatedapoptotic pathway, resulting in cell death of hyperproliferative cellsexpressing zcytor16-comprising receptors.

12. Therapeutic Uses of Polypeptides Having Zcytor16 Activity

Amino acid sequences having Zcytor16 activity can be used to modulatethe immune system by binding Zcytor16 ligand, and thus, preventing thebinding of Zcytor16 ligand with endogenous Zcytor16 receptor. Zcytor16antagonists, such as anti-Zcytor16 antibodies, can also be used tomodulate the immune system by inhibiting the binding of Zcytor16 ligandwith the endogenous Zcytor16 receptor. Accordingly, the presentinvention includes the use of proteins, polypeptides, and peptideshaving Zcytor16 activity (such as Zcytor16 polypeptides, Zcytor16analogs (e.g., anti-Zcytor16 anti-idiotype antibodies), and Zcytor16fusion proteins) to a subject which lacks an adequate amount of thispolypeptide, or which produces an excess of Zcytor16 ligand. Zcytor16antagonists (e.g., anti-Zcytor16 antibodies) can be also used to treat asubject which produces an excess of either Zcytor16 ligand or Zcytor16.Suitable subjects include mammals, such as humans.

Moreover, we have shown that the zcytor16 receptor binds a ligand calledT-cell inducible Factor (IL-TIF) (SEQ ID NO:15; Dumoutier, L. et al.,Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; mouse IL-TIF sequence isshown in Dumontier et al., J. Immunol. 164:1814-1819, 2000). Moreover,commonly owned zcytor11 (U.S. Pat. No. 5,965,704) and CRF2-4 receptoralso bind IL-TIF (See, WIPO publication WO 00/24758; Dumontier et al.,J. Immunol. 164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med.187:571-578, 1998; Gibbs, VC and Pennica Gene 186:97-101, 1997 (CRF2-4cDNA); Xie, M H et al., J. Biol. Chem. 275: 31335-31339, 2000; andKotenko, S V et al., J. Biol. Chem. manuscript in press M007837200).Moreover, IL-10β receptor may be involved as a receptor for IL-TIF, andit is believed to be synonymous with CRF2-4 (Dumoutier, L. et al., Proc.Nat'l. Acad. Sci. 97:10144-10149, 2000; Liu Y et al, J. Immunol. 152;1821-1829, 1994 (IL-10R cDNA). Within preferred embodiments, the solublereceptor form of zcytor16, residues 22-231 of SEQ ID NO:2, (SEQ IDNO:13) is a monomer, homodimer, heterodimer, or multimer thatantagonizes the effects of IL-TIF in vivo. Antibodies and bindingpolypeptides to such zcytor16 monomer, homodimer, heterodimer, ormultimers also serve as antagonists of zcytor16 activity.

IL-TIF has been shown to be induced in the presence of IL-9, and issuspected to be involved in promoting Th1-type immune responses, andinflammation. IL-9 stimulates proliferation, activation, differentiationand/or induction of immune function in a variety of ways and isimplicated in asthma, lung mastocytosis, and other diseases, as well asactivates STAT pathways. Antagonists of IL-TIF or IL-9 function can havebeneficial use against such human diseases. The present inventionprovides such novel antagonists of IL-TIF.

IL-TIF has been show to be involved in up-regulate the production ofacute phase reactants, such as serum amyloid A (SAA),α1-antichymotrypsin, and haptoglobin, and that IL-TIF expression isincreased upon injection of lipopolysaccharide (LPS) in vivo suggestingthat IL-TIF is involved in inflammatory response (Dumoutier, L. et al.,Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000). Production of acute phaseproteins, such as SAA, is considered s short-term survival mechanismwhere inflammation is beneficial; however, maintenance of acute phaseproteins for longer periods contributes to chronic inflammation and canbe harmful to human health. For review, see Uhlar, C M and Whitehead, AS, Eur. J. Biochem. 265:501-523, 1999, and Baumann H. and Gauldie, J.Immunology Today 15:74-80, 1994. Moreover, the acute phase protein SAAis implicated in the pathogenesis of several chronic inflammatorydiseases, is implicated in atherosclerosis and rheumatoid arthritis, andis the precursor to the amyloid A protein deposited in amyloidosis(Uhlar, C M and Whitehead, supra.). Thus, as IL-TIF acts as apro-inflammatory molecule and induces production of SAA, antagonistswould be useful in treating inflammatory disease and other diseasesassociated with acute phase response proteins induced by IL-TIF. Suchantagonists are provided by the present invention. For example, methodof reducing IL-TIF-induced or IL-9 induced inflammation comprisesadministering to a mammal with inflammation an amount of a compositionof soluble zcytor16-comprising receptor sufficient to reduceinflammation. Moreover, a method of suppressing an inflammatory responsein a mammal with inflammation can comprise: (1) determining a level ofserum amyloid A protein; (2) administering a composition comprising asoluble zcytor16 cytokine receptor polypeptide as described herein in anacceptable pharmaceutical vehicle; (3) determining a post administrationlevel of serum amyloid A protein; (4) comparing the level of serumamyloid A protein in step (1) to the level of serum amyloid A protein instep (3), wherein a lack of increase or a decrease in serum amyloid Aprotein level is indicative of suppressing an inflammatory response.

The receptors of the present invention include at least one zcytor16receptor subunit. A second receptor polypeptide included in theheterodimeric soluble receptor belongs to the receptor subfamily thatincludes Interleukin-10 receptor, the interferons (e.g.,interferon-gamma alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains), zcytor7, zcytor11, and CRF2-4. A secondsoluble receptor polypeptide included in a heterodimeric solublereceptor can also include a zcytor11 soluble receptor subunit, disclosedin the commonly owned U.S. Pat. No. 5,965,704; an IL-10R subunit, suchas IL-10Rα; or a zcytor7 soluble receptor subunit, disclosed in thecommonly owned U.S. Pat. No. 5,945,511. The zcytor11 receptor inconjunction with CRF2-4 and IL-10 Receptor was shown to signal JAK-STATpathway in response to IL-TIF (Xie et al., supra.; Kotenko et al.,supra.). According to the present invention, in addition to a monomericor homodimeric zcytor16 receptor polypeptide, a heterodimeric solublezcytor16 receptor, as exemplified by an embodiment comprising a solublezcytor16 receptor+soluble CRF2-4 receptor heterodimer (zcytor16/CRF2-4),can act as an antagonist of the IL-TIF. Other embodiments includesoluble heterodimers comprising zcytor16/IL-10R, zcytor16/IL-9R,zcytor16/zcytor11, zcytor16/zcytor7, and other class II receptorsubunits, as well as multimeric receptors including but not limited tozcytor16/CRF2-4/zcytor11 or zcytor16/CRF2-4/IL-10R.

Analysis of the tissue distribution of the mRNA corresponding zcytor16cDNA showed that mRNA level was highest in placenta and spleen, and theligand to which zcytor16 binds (IL-TIF) is implicated in inducinginflammatory response including induction of the acute-phase response(Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000).Thus, particular embodiments of the present invention are directedtoward use of soluble zcytor16 heterodimers as antagonists ininflammatory and immune diseases or conditions such as pancreatitis,type I diabetes (IDDM), pancreatic cancer, pancreatitis, Graves Disease,inflammatory bowel disease (IBD), Crohn's Disease, colon and intestinalcancer, diverticulosis, autoimmune disease, sepsis, organ or bone marrowtransplant; inflammation due to trauma, sugery or infection;amyloidosis; splenomegaly; graft versus host disease; and whereinhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells), suppression ofimmune response to a pathogen or antigen, or other instances whereinhibition of IL-TIF or IL-9 cytokine production is desired.

Moreover, antibodies or binding polypeptides that bind zcytor16polypeptides, monomers, homodimers, heterodimers and multimers describedherein and/or zcytor16 polypeptides, monomers, homodimers, heterodimersand multimers themselves are useful to:

1) Antagonize or block signaling via the IL-TIF receptors in thetreatment of acute inflammation, inflammation as a result of trauma,tissue injury, surgery, sepsis or infection, and chronic inflammatorydiseases such as asthma, inflammatory bowel disease (IBD), chroniccolitis, splenomegaly, rheumatoid arthritis, recurrent acuteinflammatory episodes (e.g., tuberculosis), and treatment ofamyloidosis, and atherosclerosis, Castleman's Disease, asthma, and otherdiseases associated with the induction of acute-phase response.

2) Antagonize or block signaling via the IL-TIF receptors in thetreatment of autoimmune diseases such as IDDM, multiple sclerosis (MS),systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoidarthritis, and IBD to prevent or inhibit signaling in immune cells (e.g.lymphocytes, monocytes, leukocytes) via zcytor16 (Hughes C et al., J.Immunol. 153: 3319-3325, 1994). Alternatively antibodies, such asmonoclonal antibodies (MAb) to zcytor16-comprising receptors, can alsobe used as an antagonist to deplete unwanted immune cells to treatautoimmune disease. Asthma, allergy and other atopic disease may betreated with an MAb against, for example, soluble zcytor16 solublereceptors or zcytor16/CRF2-4 heterodimers, to inhibit the immuneresponse or to deplete offending cells. Blocking or inhibiting signalingvia zcytor16, using the polypeptides and antibodies of the presentinvention, may also benefit diseases of the pancreas, kidney, pituitaryand neuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinomamay benefit. Zcytor16 may serve as a target for MAb therapy of cancerwhere an antagonizing MAb inhibits cancer growth and targetsimmune-mediated killing. (Holliger P, and Hoogenboom, H: Nature Biotech.16: 1015-1016, 1998). Mabs to soluble zcytor16 monomers, homodimers,heterodimers and multimers may also be useful to treat nephropathiessuch as glomerulosclerosis, membranous neuropathy, amyloidosis (whichalso affects the kidney among other tissues), renal arteriosclerosis,glomerulonephritis of various origins, fibroproliferative diseases ofthe kidney, as well as kidney dysfunction associated with SLE, IDDM,type II diabetes (NIDDM), renal tumors and other diseases.

3) Agonize or initiate signaling via the IL-TIF receptors in thetreatment of autoimmune diseases such as IDDM, MS, SLE, myastheniagravis, rheumatoid arthritis, and IBD. Anti-soluble zcytor16,anti-soluble zcytor16/CRF2-4 heterodimers and multimer monoclonalantibodies may signal lymphocytes or other immune cells todifferentiate, alter proliferation, or change production of cytokines orcell surface proteins that ameliorate autoimmunity. Specifically,modulation of a T-helper cell response to an alternate pattern ofcytokine secretion may deviate an autoimmune response to amelioratedisease (Smith J A et al., J. Immunol. 160:4841-4849, 1998). Similarly,agonistic Anti-soluble zcytor16, anti-soluble zcytor16/CRF2-4heterodimers and multimer monoclonal antibodies may be used to signal,deplete and deviate immune cells involved in asthma, allergy and atopoicdisease. Signaling via zcytor16 may also benefit diseases of thepancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,pancreatitis, and pancreatic carcinoma may benefit. Zcytor16 may serveas a target for MAb therapy of pancreatic cancer where a signaling MAbinhibits cancer growth and targets immune-mediated killing (Tutt, A L etal., J. Immunol. 161: 3175-3185, 1998). Similarly renal cell carcinomamay be treated with monoclonal antibodies to zcytor16-comprising solublereceptors of the present invention.

Soluble zcytor16 monomeric, homodimeric, heterodimeric and multimericpolypeptides described herein can be used to neutralize/block IL-TIFactivity in the treatment of autoimmune disease, atopic disease, NIDDM,pancreatitis and kidney dysfunction as described above. A soluble formof zcytor16 may be used to promote an antibody response mediated by Thcells and/or to promote the production of IL-4 or other cytokines bylymphocytes or other immune cells.

The soluble zcytor16-comprising receptors of the present invention areuseful as antagonists of the IL-TIF cytokine. Such antagonistic effectscan be achieved by direct neutralization or binding of the IL-TIF. Inaddition to antagonistic uses, the soluble receptors of the presentinvention can bind IL-TIF and act as carrier proteins for the IL-TIFcytokine, in order to transport the Ligand to different tissues, organs,and cells within the body. As such, the soluble receptors of the presentinvention can be fused or coupled to molecules, polypeptides or chemicalmoieties that direct the soluble-receptor-Ligand complex to a specificsite, such as a tissue, specific immune cell, or tumor. For example, inacute infection or some cancers, benefit may result from induction ofinflammation and local acute phase response proteins by the action ofIL-TIF. Thus, the soluble receptors of the present invention can be usedto specifically direct the action of the IL-TIF. See, Cosman, D.Cytokine 5: 95-106, 1993; and Fernandez-Botran, R. Exp. Opin. Invest.Drugs 9:497-513, 2000.

Moreover, the soluble receptors of the present invention can be used tostabilize the IL-TIF, to increase the bioavailability, therapeuticlongevity, and/or efficacy of the Ligand by stabilizing the Ligand fromdegradation or clearance, or by targeting the ligand to a site of actionwithin the body. For example the naturally occurring IL-6/soluble IL-6Rcomplex stabilizes IL-6 and can signal through the gp130 receptor. See,Cosman, D. supra., and Fernandez-Botran, R. supra. Moreover, Zcytor16may be combined with a cognate ligand such as IL-TIF to comprise aligand/soluble receptor complex. Such complexes may be used to stimulateresponses from cells presenting a companion receptor subunit such as,for example, zcytor11 or CRF2-4. The cell specificity of zcytor16/ligandcomplexes may differ from that seen for the ligand administered alone.Furthermore the complexes may have distinct pharmacokinetic propertiessuch as affecting half-life, dose/response and organ or tissuespecificity. ZcytoR16/IL-TIF complexes thus may have agonist activity toenhance an immune response or stimulate mesangial cells or to stimulatehepatic cells. Alternatively only tissues expressing a signaling subunitthe heterodimerizes with the complex may be affected analogous to theresponse to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l. Acad.Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.) “The CytokineHandbook”, 3^(rd) Ed., p. 208-209). Soluble receptor/cytokine complexesfor IL12 and CNTF display similar activities.

Zcytor16 homodimeric, heterodimeric and multimeric receptor polypeptidesmay also be used within diagnostic systems for the detection ofcirculating levels of IL-TIF ligand, and in the detection of IL-TIFassociated with acute phase inflammatory response. Within a relatedembodiment, antibodies or other agents that specifically bind toZcytor16 soluble receptors of the present invention can be used todetect circulating receptor polypeptides; conversely, Zcytor16 solublereceptors themselves can be used to detect circulating or locally-actingIL-TIF polypeptides. Elevated or depressed levels of ligand or receptorpolypeptides may be indicative of pathological conditions, includinginflammation or cancer. IL-TIF is known to induce associated acute phaseinflammatory response. Moreover, detection of acute phase proteins ormolecules such as IL-TIF can be indicative of a chronic inflammatorycondition in certain disease states (e.g., rheumatoid arthritis).Detection of such conditions serves to aid in disease diagnosis as wellas help a physician in choosing proper therapy.

Moreover, soluble zcytor16 receptor polypeptides of the presentinvention can be used as a “ligand sink,” i.e., antagonist, to bindligand in vivo or in vitro in therapeutic or other applications wherethe presence of the ligand is not desired. For example, in chronicinflammatory conditions or cancers that are expressing large amounts ofbioactive IL-TIF, soluble zcytor16 receptor or soluble zcytor16heterodimeric and multimeric receptor polypeptides, such as solublezcytor16/CRF2-4 can be used as a direct antagonist of the ligand invivo, and may aid in reducing progression and symptoms associated withthe disease, and can be used in conjunction with other therapies (e.g.,steroid or chemotherapy) to enhance the effect of the therapy inreducing progression and symptoms, and preventing relapse. Moreover,soluble zcytor16 receptor polypeptides can be used to slow theprogression of cancers that over-express zcytor16 receptors, by bindingligand in vivo that could otherwise enhance proliferation of thosecancers.

Moreover, soluble zcytor16 receptor polypeptides of the presentinvention can be used in vivo or in diagnostic applications to detectIL-TIF-expressing inflammation or cancers in vivo or in tissue samples.For example, the soluble zcytor16 receptors of the present invention canbe conjugated to a radio-label or fluorescent label as described herein,and used to detect the presence of the IL-TIF in a tissue sample usingan in vitro ligand-receptor type binding assay, or fluorescent imagingassay. Moreover, radiolabeled soluble zcytor16 receptors of the presentinvention could be administered in vivo to detect Ligand-expressingsolid tumors through a radio-imaging method known in the art.

Generally, the dosage of administered Zcytor16 (or Zcytor16 analog orfusion protein) will vary depending upon such factors as the patient'sage, weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of Zcytor16 polypeptide which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate.

Administration of a Zcytor16 polypeptide to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. Drug Dev.Rev. 35:199 (1999)). Dry or liquid particles comprising Zcytor16 can beprepared and inhaled with the aid of dry-powder dispersers, liquidaerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH16:343 (1998); Patton et al, Adv. Drug Deliv. Rev. 35:235 (1999)). Thisapproach is illustrated by the AERX diabetes management system, which isa hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al, Science 269:850 (1995)).Transdermal delivery using electroporation provides another means toadminister a molecule having Zcytor16 binding activity (Potts et al,Pharm. Biotechnol 10:213 (1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zcytor16 binding activity can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe therapeutic proteins are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having Zcytor16 binding activity anda pharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having Zcytor16 binding activity and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse.

A pharmaceutical composition comprising Zcytor16 (or Zcytor16 analog orfusion protein) can be furnished in liquid form, in an aerosol, or insolid form. Liquid forms, are illustrated by injectable solutions andoral suspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997);Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al, Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al, American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al, Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal, Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al, Biochim. Biophys.Acta 1068: 133 (1991); Allen et al, Biochim. Biophys. Acta 1150:9(1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al, Japanese Patent 04-244,018; Kato et al., Biol Pharm. Bul116:960(1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al, Biol Pharm. Bull 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al, Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al, Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al, Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al, Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al, Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al, Adv. Drug Deliv. Rev.32:99 (1998)).

Polypeptides having Zcytor16 binding activity can be encapsulated withinliposomes using standard techniques of protein microencapsulation (see,for example, Anderson et al, Infect. Immun. 31:1099 (1981), Anderson etal, Cancer Res. 50:1853 (1990), and Cohen et al, Biochim. Biophys. Acta1063:95 (1991), Alving et al “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al, Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al, Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving binding Zcytor16 activity such as zcytor16 monomeric,homodimeric, heterodimeric or multimeric soluble receptors, and Zcytor16antagonists, for example anti-zcytor16 antibodies or bindingpolypeptides, which a polypeptide is linked with a polymer, as discussedabove.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a polypeptide with a Zcytor16extracellular domain, e.g., zcytor16 monomeric, homodimeric,heterodimeric or multimeric soluble receptors, or a Zcytor16 antagonist(e.g., an antibody or antibody fragment that binds a Zcytor16polypeptide). Therapeutic polypeptides can be provided in the form of aninjectable solution for single or multiple doses, or as a sterile powderthat will be reconstituted before injection. Alternatively, such a kitcan include a dry-powder disperser, liquid aerosol generator, ornebulizer for administration of a therapeutic polypeptide. Such a kitmay further comprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the Zcytor16 composition is contraindicated in patientswith known hypersensitivity to Zcytor16.

13. Therapeutic Uses of Zcytor16 Nucleotide Sequences

The present invention includes the use of Zcytor16 nucleotide sequencesto provide Zcytor16 to a subject in need of such treatment. In addition,a therapeutic expression vector can be provided that inhibits Zcytor16gene expression, such as an anti-sense molecule, a ribozyme, or anexternal guide sequence molecule.

There are numerous approaches to introduce a Zcytor16 gene to a subject,including the use of recombinant host cells that express Zcytor16,delivery of naked nucleic acid encoding Zcytor16, use of a cationiclipid carrier with a nucleic acid molecule that encodes Zcytor16, andthe use of viruses that express Zcytor16, such as recombinantretroviruses, recombinant adeno-associated viruses, recombinantadenoviruses, and recombinant Herpes simplex viruses (see, for example,Mulligan, Science 260:926 (1993), Rosenberg et al, Science 242:1575(1988), LaSalle et al., Science 259:988 (1993), Wolff et al., Science247:1465 (1990), Breakfield and Deluca, The New Biologist 3:203 (1991)).In an ex vivo approach, for example, cells are isolated from a subject,transfected with a vector that expresses a Zcytor16 gene, and thentransplanted into the subject.

In order to effect expression of a Zcytor16 gene, an expression vectoris constructed in which a nucleotide sequence encoding a Zcytor16 geneis operably linked to a core promoter, and optionally a regulatoryelement, to control gene transcription. The general requirements of anexpression vector are described above.

Alternatively, a Zcytor16 gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal, Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Nat'lAcad. Sci. USA 91:215 (1994), Li et al, Hum. Gene Ther. 4:403 (1993),Vincent et al, Nat. Genet. 5:130 (1993), and Zabner et al, Cell 75:207(1993)), adenovirus-associated viral vectors (Flotte et al, Proc. Nat'lAcad. Sci. USA 90:10613 (1993)), alphaviruses such as Semliki ForestVirus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857 (1992), Rajuand Huang, J. Vir. 65:2501 (1991), and Xiong et al, Science 243:1188(1989)), herpes viral vectors (e.g., U.S. Pat. Nos. 4,769,331,4,859,587, 5,288,641 and 5,328,688), parvovirus vectors (Koering et al.,Hum. Gene Therap. 5:457 (1994)), pox virus vectors (Ozaki et al,Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali and Paoletti,Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, such as canarypox virus or vaccinia virus (Fisher-Hoch et al, Proc. Nat'l Acad. Sci.USA 86:317 (1989), and Flexner et al, Ann. N.Y. Acad. Sci. 569:86(1989)), and retroviruses (e.g., Baba et al, J. Neurosurg 79:729 (1993),Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J. Neurosci. Res33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993), Vile and Hart,Cancer Res. 53:3860 (1993), and Anderson et al, U.S. Pat. No.5,399,346). Within various embodiments, either the viral vector itself,or a viral particle which contains the viral vector may be utilized inthe methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al,Meth. Cell Biol 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al, J. Virol 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al, J. Virol 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant herpes simplex virus can beprepared in Vero cells, as described by Brandt et al, J. Gen. Virol.72:2043 (1991), Herold et al, J. Gen. Virol 75:1211 (1994), Visalli andBrandt, Virology 185:419 (1991), Grau et al, Invest. Ophthalmol. Vis.Sci. 30:2474 (1989), Brandt et al, J. Virol Meth. 36:209 (1992), and byBrown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).

Alternatively, an expression vector comprising a Zcytor16 gene can beintroduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al, Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

Electroporation is another alternative mode of administration. Forexample, Aihara and Miyazaki, Nature Biotechnology 16:867 (1998), havedemonstrated the use of in vivo electroporation for gene transfer intomuscle.

In an alternative approach to gene therapy, a therapeutic gene mayencode a Zcytor16 anti-sense RNA that inhibits the expression ofZcytor16. Suitable sequences for anti-sense molecules can be derivedfrom the nucleotide sequences of Zcytor16 disclosed herein.

Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in a mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zcytor16 mRNA.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode aZcytor16 gene. According to this approach, an external guide sequencecan be constructed for directing the endogenous ribozyme, RNase P, to aparticular species of intracellular mRNA, which is subsequently cleavedby the cellular ribozyme (see, for example, Altman et al, U.S. Pat. No.5,168,053, Yuan et al, Science 263:1269 (1994), Pace et al,international publication No. WO 96/18733, George et al, internationalpublication No. WO 96/21731, and Werner et al, international publicationNo. WO 97/33991). For example, the external guide sequence can comprisea ten to fifteen nucleotide sequence complementary to Zcytor16 mRNA, anda 3′-NCCA nucleotide sequence, wherein N is preferably a purine. Theexternal guide sequence transcripts bind to the targeted mRNA species bythe formation of base pairs between the mRNA and the complementaryexternal guide sequences, thus promoting cleavage of mRNA by RNase P atthe nucleotide located at the 5′-side of the base-paired region.

In general, the dosage of a composition comprising a therapeutic vectorhaving a Zcytor16 nucleotide sequence, such as a recombinant virus, willvary depending upon such factors as the subject's age, weight, height,sex, general medical condition and previous medical history. Suitableroutes of administration of therapeutic vectors include intravenousinjection, intraarterial injection, intraperitoneal injection,intramuscular injection, intratumoral injection, and injection into acavity that contains a tumor. As an illustration, Horton et al, Proc.Nat'l Acad. Sci. USA 96:1553 (1999), demonstrated that intramuscularinjection of plasmid DNA encoding interferon-α produces potent antitumoreffects on primary and metastatic tumors in a murine model.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

14. Production of Transgenic Mice

Transgenic mice can be engineered to over-express the Zcytor16 gene inall tissues or under the control of a tissue-specific ortissue-preferred regulatory element. These over-producers of Zcytor16can be used to characterize the phenotype that results fromover-expression, and the transgenic animals can serve as models forhuman disease caused by excess Zcytor16. Transgenic mice thatover-express Zcytor16 also provide model bioreactors for production ofZcytor16, such as soluble Zcytor16, in the milk or blood of largeranimals. Methods for producing transgenic mice are well-known to thoseof skill in the art (see, for example, Jacob, “Expression and Knockoutof Interferons in Transgenic Mice,” in Overexpression and Knockout ofCytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (AcademicPress, Ltd. 1994), Monastersky and Robl (eds.), Strategies in TransgenicAnimal Science (ASM Press 1995), and Abbud and Nilson, “RecombinantProtein Expression in Transgenic Mice,” in Gene Expression Systems:Using Nature for the Art of Expression, Fernandez and Hoeffler (eds.),pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses aZcytor16 gene can begin with adult, fertile males (studs) (B6C3f1, 2-8months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males(duds) (B6D2f1, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2f1, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a Zcytor16 encodingsequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10nanograms per microliter for microinjection. For example, the Zcytor16encoding sequences can encode a polypeptide comprising the amino acidsequence of SEQ ID NO:2.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a Zcytor16 gene or aselectable marker gene that was introduced in the same plasmid. Afteranimals are confirmed to be transgenic, they are back-crossed into aninbred strain by placing a transgenic female with a wild-type male, or atransgenic male with one or two wild-type female(s). As pups are bornand weaned, the sexes are separated, and their tails snipped forgenotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zcytor16 mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

In addition to producing transgenic mice that over-express Zcytor16, itis useful to engineer transgenic mice with either abnormally low or noexpression of the gene. Such transgenic mice provide useful models fordiseases associated with a lack of Zcytor16. As discussed above,Zcytor16 gene expression can be inhibited using anti-sense genes,ribozyme genes, or external guide sequence genes. To produce transgenicmice that under-express the Zcytor16 gene, such inhibitory sequences aretargeted to Zcytor16 mRNA. Methods for producing transgenic mice thathave abnormally low expression of a particular gene are known to thosein the art (see, for example, Wu et al, “Gene Underexpression inCultured Cells and Animals by Antisense DNA and RNA Strategies,” inMethods in Gene Biotechnology, pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno Zcytor16 gene expression is to generate mice having at least onenormal Zcytor16 allele replaced by a nonfunctional Zcytor16 gene. Onemethod of designing a nonfunctional Zcytor16 gene is to insert anothergene, such as a selectable marker gene, within a nucleic acid moleculethat encodes Zcytor16. Standard methods for producing these so-called“knockout mice” are known to those skilled in the art (see, for example,Jacob, “Expression and Knockout of Interferons in Transgenic Mice,” inOverexpression and Knockout of Cytokines in Transgenic Mice, Jacob(ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al., “NewStrategies for Gene Knockout,” in Methods in Gene Biotechnology, pages339-365 (CRC Press 1997)).

Polynucleotides and polypeptides of the present invention willadditionally find use as educational tools as a laboratory practicumkits for courses related to genetics and molecular biology, proteinchemistry and antibody production and analysis. Due to its uniquepolynucleotide and polypeptide sequence molecules of zcytor16 can beused as standards or as “unknowns” for testing purposes. For example,zcytor16 polynucleotides can be used as an aid, such as, for example, toteach a student how to prepare expression constructs for bacterial,viral, and/or mammalian expression, including fusion constructs, whereinzcytor16 is the gene to be expressed; for determining the restrictionendonuclease cleavage sites of the polynucleotides; determining mRNA andDNA localization of zcytor16 polynucleotides in tissues (i.e., byNorthern and Southern blotting as well as polymerase chain reaction);and for identifying related polynucleotides and polypeptides by nucleicacid hybridization.

Zcytor16 polypeptides can be used educationally as an aid to teachpreparation of antibodies; identifying proteins by Western blotting;protein purification; determining the weight of expressed zcytor16polypeptides as a ratio to total protein expressed; identifying peptidecleavage sites; coupling amino and carboxyl terminal tags; amino acidsequence analysis, as well as, but not limited to monitoring biologicalactivities of both the native and tagged protein (i.e., receptorbinding, signal transduction, proliferation, and differentiation) invitro and in vivo. Zcytor16 polypeptides can also be used to teachanalytical skills such as mass spectrometry, circular dichroism todetermine conformation, especially of the four alpha helices, x-raycrystallography to determine the three-dimensional structure in atomicdetail, nuclear magnetic resonance spectroscopy to reveal the structureof proteins in solution. For example, a kit containing the zcytor16 canbe given to the student to analyze. Since the amino acid sequence wouldbe known by the professor, the specific protein can be given to thestudent as a test to determine the skills or develop the skills of thestudent, the teacher would then know whether or not the student hascorrectly analyzed the polypeptide. Since every polypeptide is unique,the educational utility of zcytor16 would be unique unto itself.

Moreover, since zcytor16 has a tissue-specific expression and is apolypeptide with a class II cytokine receptor structure and a distinctchromosomal localization, and expressin pattern, activity can bemeasured using proliferation assays; luciferase and binding assaysdescribed herein. Moreover, expression of zcytor16 polynucleotides andpolypeptides in lymphoid and other tissues can be analyzed in order totrain students in the use of diagnostic and tissue-specificidentification and methods. Moreover zcytor16 polynucleotides can beused to train students on the use of chromosomal detection anddiagnostic methods, since it's locus is known. Moreover, students can bespecifically trained and educated about human chromosome 1, and morespecifically the locus 6q24.1-25.2 wherein the zcytor16 gene islocalized. Such assays are well known in the art, and can be used in aneducational setting to teach students about cytokine receptor proteinsand examine different properties, such as cellular effects on cells,enzyme kinetics, varying antibody binding affinities, tissuespecificity, and the like, between zcytor16 and other cytokine receptorpolypeptides in the art.

The antibodies which bind specifically to zcytor16 can be used as ateaching aid to instruct students how to prepare affinity chromatographycolumns to purify zcytor16, cloning and sequencing the polynucleotidethat encodes an antibody and thus as a practicum for teaching a studenthow to design humanized antibodies. Moreover, antibodies which bindspecifically to zcytor16 can be used as a teaching aid for use indetection e.g., of activated CD91+ cells, cell sorting, or ovariancancer tissue using histological, and in situ methods amongst othersknown in the art. The zcytor16 gene, polypeptide or antibody would thenbe packaged by reagent companies and sold to universities and othereducational entities so that the students gain skill in art of molecularbiology. Because each gene and protein is unique, each gene and proteincreates unique challenges and learning experiences for students in a labpracticum. Such educational kits containing the zcytor16 gene,polypeptide or antibody are considered within the scope of the presentinvention.

Within one aspect the present invention provides an isolatedpolypeptide, comprising at least 15 contiguous amino acid residues of anamino acid sequence of SEQ ID NO:2 selected from the group consistingof: (a) amino acid residues amino acid residues 21 to 231, (b) aminoacid residues 21 to 210, (c) amino acid residues 22 to 231, (d) aminoacid residues 22 to 210, (e) amino acid residues 22 to 108, (f) aminoacid residues 112 to 210, and (g) amino acid residues 21 to 110. In oneembodiment, the isolated polypeptide disclosed above comprises an aminoacid sequence selected from the group consisting of: (a) amino acidresidues amino acid residues 21 to 231, (b) amino acid residues 21 to210, (c) amino acid residues 22 to 231, (d) amino acid residues 22 to210, (e) amino acid residues 22 to 108, (f) amino acid residues 112 to210, and (g) amino acid residues 21 to 110. In another embodiment, theisolated polypeptide disclosed above consists of an amino acid sequenceselected from the group consisting of: (a) amino acid residues aminoacid residues 21 to 231, (b) amino acid residues 21 to 210, (c) aminoacid residues 22 to 231, (d) amino acid residues 22 to 210, (e) aminoacid residues 22 to 108, (f) amino acid residues 112 to 210, and (g)amino acid residues 21 to 110.

Within a second aspect the present invention provides an isolatedpolypeptide, comprising an amino acid sequence that is at least 70%identical to a reference amino acid sequence of SEQ ID NO:2 selectedfrom the group consisting of: (a) amino acid residues amino acidresidues 21 to 231, (b) amino acid residues 21 to 210, (c) amino acidresidues 22 to 231, (d) amino acid residues 22 to 210, (e) amino acidresidues 22 to 108, (f) amino acid residues 112 to 210, and (g) aminoacid residues 21 to 110. In one embodiment, the isolated polypeptidedisclosed above has an amino acid sequence that is at least 80%identical to the reference amino acid sequence. In another embodiment,the isolated polypeptide disclosed above has an amino acid sequence thatis at least 90% identical to the reference amino acid sequence. Inanother embodiment, the isolated polypeptide disclosed above compriseseither amino acid residues 22 to 231 of SEQ ID NO:2 or amino acidresidues 22 to 210 of SEQ ID NO:2.

Within a third aspect the present invention provides an isolated nucleicacid molecule, wherein the nucleic acid molecule is either (a) a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:3, or (b)a nucleic acid molecule that remains hybridized following stringent washconditions to a nucleic acid molecule consisting of the nucleotidesequence of nucleotides 64 to 630 of SEQ ID NO:1, or the complement ofthe nucleotide sequence of nucleotides 64 to 630 of SEQ ID NO:1. In oneembodiment, the isolated nucleic acid molecule is as disclosed abovewherein any difference between the amino acid sequence encoded by thenucleic acid molecule and the corresponding amino acid sequence of SEQID NO:2 is due to a conservative amino acid substitution. In anotherembodiment, the isolated nucleic acid molecule disclosed above comprisesthe nucleotide sequence of nucleotides 64 to 630 of SEQ ID NO:1.

Within another aspect the present invention provides a vector,comprising the isolated nucleic acid molecule as disclosed above.

Within another aspect the present invention provides an expressionvector, comprising the isolated nucleic acid molecule as disclosedabove, a transcription promoter, and a transcription terminator, whereinthe promoter is operably linked with the nucleic acid molecule, andwherein the nucleic acid molecule is operably linked with thetranscription terminator.

Within another aspect the present invention provides a recombinant hostcell comprising the expression vector as disclosed above, wherein thehost cell is selected from the group consisting of bacterium, yeastcell, fungal cell, insect cell, mammalian cell, and plant cell.

Within another aspect the present invention provides a method ofproducing Zcytor16 protein, the method comprising culturing recombinanthost cells that comprise the expression vector as disclosed above, andthat produce the Zcytor16 protein. In one embodiment, the methoddisclosed above, further comprises isolating the Zcytor16 protein fromthe cultured recombinant host cells.

Within another aspect the present invention provides an antibody orantibody fragment that specifically binds with the polypeptide asdisclosed above. In one embodiment, the antibody disclosed above isselected from the group consisting of: (a) polyclonal antibody, (b)murine monoclonal antibody, (c) humanized antibody derived from (b), and(d) human monoclonal antibody.

Within another aspect the present invention provides an anti-idiotypeantibody that specifically binds with the antibody as disclosed above.

Within another aspect the present invention provides a fusion protein,comprising the polypeptide as disclosed above. In one embodiment, thefusion protein disclosed above further comprises an immunoglobulinmoiety.

Within another aspect the present invention provides an isolatedpolynucleotide that encodes a soluble cytokine receptor polypeptidecomprising a sequence of amino acid residues that is at least 90%identical to the amino acid sequence as shown in SEQ ID NO:2 from aminoacid 22-231 or 22-210, and wherein the soluble cytokine receptorpolypeptide encoded by the polynucleotide sequence binds IL-TIF orantagonizes IL-TIF activity.

Within another aspect the present invention provides an isolatedpolynucleotide as disclosed above, wherein the soluble cytokine receptorpolypeptide encoded by the polynucleotide forms a homodimeric,heterodimeric or multimeric receptor complex. In one embodiment, theisolated polynucleotide is as disclosed above, wherein the solublecytokine receptor polypeptide encoded by the polynucleotide forms aheterodimeric or multimeric receptor complex further comprising asoluble Class I or Class II cytokine receptor. In another embodiment,the isolated polynucleotide is as disclosed above, wherein the solublecytokine receptor polypeptide encoded by the polynucleotide forms aheterodimeric or multimeric receptor complex further comprising asoluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble IL-10receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptorpolypeptide (SEQ ID NO:34).

Within another aspect the present invention provides an isolatedpolynucleotide that encodes a soluble cytokine receptor polypeptidecomprising a sequence of amino acid residues as shown in SEQ ID NO:2from amino acid 22-231 or 22-210, wherein the soluble cytokine receptorpolypeptide encoded by the polynucleotide forms a homodimeric,heterodimeric or multimeric receptor complex. In one embodiment, theisolated polynucleotide is as disclosed above, wherein the solublecytokine receptor polypeptide encoded by the polynucleotide furthercomprises a soluble Class I or Class II cytokine receptor. In anotherembodiment, the isolated polynucleotide is as disclosed above, whereinthe soluble cytokine receptor polypeptide encoded by the polynucleotideforms a heterodimeric or multimeric receptor complex further comprisinga soluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble IL-10receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptorpolypeptide (SEQ ID NO:34). In another embodiment, the isolatedpolynucleotide is as disclosed above, wherein the soluble cytokinereceptor polypeptide further encodes an intracellular domain. In anotherembodiment, the isolated polynucleotide is as disclosed above, whereinthe soluble cytokine receptor polypeptide further comprises an affinitytag.

Within another aspect the present invention provides an expressionvector comprising the following operably linked elements: (a) atranscription promoter; a first DNA segment encoding a soluble cytokinereceptor polypeptide having an amino acid sequence as shown in SEQ IDNO:2 from amino acid 22-231 or 22-210; and a transcription terminator;and (b) a second transcription promoter; a second DNA segment encoding asoluble Class I or Class II cytokine receptor polypeptide; and atranscription terminator; and wherein the first and second DNA segmentsare contained within a single expression vector or are contained withinindependent expression vectors. In one embodiment, the expression vectordisclosed above further comprising a secretory signal sequence operablylinked to the first and second DNA segments. In another embodiment, theexpression vector is as disclosed above, wherein the second DNA segmentencodes a polypeptide comprising a soluble CRF2-4 receptor polypeptide(SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), orsoluble zcytor11 receptor polypeptide (SEQ ID NO:34).

Within another aspect the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the cellexpresses the polypeptides encoded by the DNA segments.

Within another aspect the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the firstand second DNA segments are located on independent expression vectorsand are co-transfected into the cell, and cell expresses thepolypeptides encoded by the DNA segments.

Within another aspect the present invention provides a cultured cellinto which has been introduced an expression vector as disclosed above,wherein the cell expresses a heterodimeric or multimeric solublereceptor polypeptide encoded by the DNA segments. In one embodiment isprovided a cell as disclosed above, wherein the cell secretes a solublecytokine receptor polypeptide heterodimer or multimeric complex. Inanother embodiment is provided a cell as disclosed above, wherein thecell secretes a soluble cytokine receptor polypeptide heterodimer ormultimeric complex that binds IL-TIF or antagonizes IL-TIF activity.

Within another aspect the present invention provides a DNA constructencoding a fusion protein comprising: a first DNA segment encoding apolypeptide having a sequence of amino acid residues as shown in SEQ IDNO:2 from amino acid 22-231 or 22-210; and at least one other DNAsegment encoding a soluble Class I or Class II cytokine receptorpolypeptide, wherein the first and other DNA segments are connectedin-frame; and wherein the first and other DNA segments encode the fusionprotein. In one embodiment the DNA construct encoding a fusion proteinis as disclosed above, wherein at least one other DNA segment encodes apolypeptide comprising a soluble CRF2-4 receptor polypeptide (SEQ IDNO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), or solublezcytor11 receptor polypeptide (SEQ ID NO:34).

Within another aspect the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA construct encoding a fusion protein asdisclosed above; and a transcription terminator, wherein the promoter isoperably linked to the DNA construct, and the DNA construct is operablylinked to the transcription terminator.

Within another aspect the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the cellexpresses a polypeptide encoded by the DNA construct.

Within another aspect the present invention provides a method ofproducing a fusion protein comprising: culturing a cell as disclosedabove; and isolating the polypeptide produced by the cell.

Within another aspect the present invention provides an isolated solublecytokine receptor polypeptide comprising a sequence of amino acidresidues that is at least 90% identical to an amino acid sequence asshown in SEQ ID NO:2 from amino acid 22-231 or 22-210, and wherein thesoluble cytokine receptor polypeptide binds IL-TIF or antagonizes IL-TIFactivity.

Within another aspect the present invention provides an isolatedpolypeptide as disclosed above, wherein the soluble cytokine receptorpolypeptide forms a homodimeric, heterodimeric or multimeric receptorcomplex. In one embodiment, the isolated polypeptide is as disclosedabove, wherein the soluble cytokine receptor polypeptide forms aheterodimeric or multimeric receptor complex further comprising asoluble Class I or Class II cytokine receptor. In another embodiment,the isolated polypeptide is as disclosed above, wherein the solublecytokine receptor polypeptide forms a heterodimeric or multimericreceptor complex further comprising a soluble CRF2-4 receptorpolypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ IDNO:36), or soluble zcytor11 receptor polypeptide (SEQ ID NO:34).

Within another aspect the present invention provides an isolated solublecytokine receptor polypeptide comprising a sequence of amino acidresidues as shown in SEQ ID NO:2 from amino acid 22-231 or 22-210,wherein the soluble cytokine receptor polypeptide forms a homodimeric,heterodimeric or multimeric receptor complex. In one embodiment, theisolated soluble cytokine receptor polypeptide is as disclosed above,wherein the soluble cytokine receptor polypeptide forms a heterodimericor multimeric receptor complex further comprising a soluble Class I orClass II cytokine receptor. In another embodiment, the isolated solublecytokine receptor polypeptide is as disclosed above, wherein the solublecytokine receptor polypeptide forms a heterodimeric or multimericreceptor complex comprising a soluble CRF2-4 receptor polypeptide (SEQID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), orsoluble zcytor11 receptor polypeptide (SEQ ID NO:34). In anotherembodiment, the isolated soluble cytokine receptor polypeptide is asdisclosed above, wherein the soluble cytokine receptor polypeptidefurther comprises an affinity tag, chemical moiety, toxin, or label.

Within another aspect the present invention provides an isolatedheterodimeric or multimeric soluble receptor complex comprising solublereceptor subunits, wherein at least one of the soluble receptor subunitscomprises a soluble cytokine receptor polypeptide comprising a sequenceof amino acid residues as shown in SEQ ID NO:2 from amino acid 22-231 or22-210. In one embodiment, the isolated heterodimeric or multimericsoluble receptor complex disclosed above, further comprises a solubleClass I or Class II cytokine receptor polypeptide. In anotherembodiment, the isolated heterodimeric or multimeric soluble receptorcomplex disclosed above, further comprises a soluble CRF2-4 receptorpolypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ IDNO:36), or soluble zcytor11 receptor polypeptide (SEQ ID NO:34).

Within another aspect the present invention provides a method ofproducing a soluble cytokine receptor polypeptide that form aheterodimeric or multimeric complex comprising: culturing a cell asdisclosed above, and isolating the soluble receptor polypeptidesproduced by the cell.

Within another aspect the present invention provides a method ofproducing an antibody to soluble cytokine receptor polypeptidecomprising: inoculating an animal with a soluble cytokine receptorpolypeptide selected from the group consisting of: (a) a polypeptidecomprising a monomeric or homodimeric soluble cytokine receptorcomprising a polypeptide as shown in SEQ ID NO:2 from amino acid 22-231or 22-210; (b) a polypeptide of (a) further comprising a solublecytokine receptor heterodimeric or multimeric receptor complexcomprising a soluble Class I or Class II cytokine receptor polypeptide;(c) a polypeptide of (a) further comprising a soluble cytokine receptorheterodimeric or multimeric receptor complex comprising a soluble CRF2-4receptor polypeptide (SEQ ID NO:35); (d) a polypeptide of (a) furthercomprising a soluble cytokine receptor heterodimeric or multimericreceptor complex comprising a soluble IL-10 receptor polypeptide (SEQ IDNO:36); and wherein the polypeptide elicits an immune response in theanimal to produce the antibody; and isolating the antibody from theanimal.

Within another aspect the present invention provides an antibodyproduced by the method as disclosed above, which specifically binds to ahomodimeric, heterodimeric or multimeric receptor complex comprising apolypeptide as shown in SEQ ID NO:2 from amino acid 22-231 or 22-210. Inone embodiment, the antibody disclosed above is a monoclonal antibody.

Within another aspect the present invention provides an antibody whichspecifically binds to a homodimeric, heterodimeric or multimericreceptor complex as disclosed above.

Within another aspect the present invention provides a method forinhibiting IL-TIF-induced proliferation or differentiation ofhematopoietic cells and hematopoietic cell progenitors comprisingculturing bone marrow or peripheral blood cells with a compositioncomprising an amount of soluble cytokine receptor polypeptide as shownin SEQ ID NO:2 from amino acid 22-231 or 22-210, sufficient to reduceproliferation or differentiation of the hematopoietic cells in the bonemarrow or peripheral blood cells as compared to bone marrow orperipheral blood cells cultured in the absence of soluble cytokinereceptor. In one embodiment, the method is as disclosed above, whereinthe hematopoietic cells and hematopoietic progenitor cells are lymphoidcells. In another embodiment, the method is as disclosed above, whereinthe lymphoid cells are macrophages or T cells.

Within another aspect the present invention provides a method ofreducing IL-TIF-induced or IL-9 induced inflammation comprisingadministering to a mammal with inflammation an amount of a compositionof a polypeptide as shown in SEQ ID NO:2 from amino acid 22-231 or22-210 sufficient to reduce inflammation.

Within another aspect the present invention provides a method ofsuppressing an inflammatory response in a mammal with inflammationcomprising: (1) determining a level of serum amyloid A protein; (2)administering a composition comprising a soluble zcytor16 cytokinereceptor polypeptide as disclosed above in an acceptable pharmaceuticalvehicle; (3) determining a post administration level of serum amyloid Aprotein; (4) comparing the level of serum amyloid A protein in step (1)to the level of serum amyloid A protein in step (3), wherein a lack ofincrease or a decrease in serum amyloid A protein level is indicative ofsuppressing an inflammatory response.

Within another aspect the present invention provides a method fordetecting a genetic abnormality in a patient, comprising: obtaining agenetic sample from a patient; producing a first reaction product byincubating the genetic sample with a polynucleotide comprising at least14 contiguous nucleotides of SEQ ID NO:1 or the complement of SEQ IDNO:1, under conditions wherein said polynucleotide will hybridize tocomplementary polynucleotide sequence; visualizing the first reactionproduct; and comparing said first reaction product to a control reactionproduct from a wild type patient, wherein a difference between saidfirst reaction product and said control reaction product is indicativeof a genetic abnormality in the patient.

Within another aspect the present invention provides a method fordetecting a cancer in a patient, comprising: obtaining a tissue orbiological sample from a patient; incubating the tissue or biologicalsample with an antibody as disclosed above under conditions wherein theantibody binds to its complementary polypeptide in the tissue orbiological sample; visualizing the antibody bound in the tissue orbiological sample; and comparing levels of antibody bound in the tissueor biological sample from the patient to a normal control tissue orbiological sample, wherein an increase in the level of antibody bound tothe patient tissue or biological sample relative to the normal controltissue or biological sample is indicative of a cancer in the patient.

Within another aspect the present invention provides a method fordetecting a cancer in a patient, comprising: obtaining a tissue orbiological sample from a patient; labeling a polynucleotide comprisingat least 14 contiguous nucleotides of SEQ ID NO:1 or the complement ofSEQ ID NO:1; incubating the tissue or biological sample with underconditions wherein the polynucleotide will hybridize to complementarypolynucleotide sequence; visualizing the labeled polynucleotide in thetissue or biological sample; and comparing the level of labeledpolynucleotide hybridization in the tissue or biological sample from thepatient to a normal control tissue or biological sample, wherein anincrease in the labeled polynucleotide hybridization to the patienttissue or biological sample relative to the normal control tissue orbiological sample is indicative of a cancer in the patient.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Cloning of Zcytor16 and Construction of MammalianExpression Vectors That Express zcytor16 Soluble Receptors: zcytor16CEE,zcytor16CFLG zcytor16CHIS and zcytor16-Fc4

A. Cloning of Zcytor16 Extracellular Domain

Scanning of a translated human genomic database resulted inidentification of a class II cytokine receptor named zcytor16. Thesequence for zcytor16 was subsequently identified a clone from anin-house derived shallow tonsil library. The insert in the tonsillibrary clone was sequenced, and shown to encode the zcytor16extracellular domain. The polynucleotide sequence of the zcytor16 cloneis shown in SEQ ID NO:1 and polypeptide sequence shown in SEQ ID NO:2.

B. Mammalian Expression Construction of Soluble Zcytor16 ReceptorZcytor16-Fc4 Construction of Mammalian Expression Vectors that Expresszcytor16 Soluble Receptor Zcytor16sR/Fc4

An expression vector was prepared to express the soluble zcytor16polypeptide (zcytor16sR, i.e., from residue 22 (Thr) to residue 231(Pro) of SEQ ID NO:2; SEQ ID NO:13) fused to a C-terminal Fc4 tag (SEQID NO:5).

A PCR generated zcytor16 DNA fragment of about 630 bp was created usingoligo ZC29,181 (SEQ ID NO:6) and oligo ZC29,182 (SEQ ID NO:7) as PCRprimers to add BamHI and Bgl2 restriction sites at 5′ and 3′ endsrespectively, of the zcytor16 DNA encoding the soluble receptor. Aplasmid containing the zcytor16 cDNA (SEQ ID NO:1) (Example 1A) was usedas a template. PCR amplification of the zcytor16 fragment was performedas follows: One cycle at 94° C. for 1 minute; 25 cycles at 94° C. for 30seconds, 68° C. for 90 seconds, followed by an additional 68° C.incubation for 4 minutes, and hold at 10° C. The reaction was purifiedby chloroform/phenol extraction and isopropanol precipitation, anddigested with BamHI and Bgl2 (Boehringer Mannheim, Indianapolis, Ind.).A band of approximately 630 bp, as visualized by 1% agarose gelelectrophoresis, was excised and the DNA was purified using a QiaexII™purification kit (Qiagen, Valencia, Calif.) according to themanufacturer's instruction.

The Fc4/pzmp20 plasmid is a mammalian expression vector containing anexpression cassette having the CMV promoter, human tPA leader peptide,multiple restriction sites for insertion of coding sequences, a Fc4 tag,and a human growth hormone terminator. The plasmid also has an E. coliorigin of replication, a mammalian selectable marker expression unithaving an SV40 promoter, an enhancer and an origin of replication, aswell as a DHFR gene, and SV40 terminator. The zcytor16sR/Fc4/pzmp20expression vector uses the human tPA leader peptide (SEQ ID NO:8 and SEQID NO:9) and attaches the Fc4 tag (SEQ ID NO:5) to the C-terminus of theextracellular portion of the zcytor16 polypeptide sequence. Fc4 is theFc region derived from human IgG, which contains a mutation so that itno longer binds the Fc receptor

About 30 ng of the restriction digested zcytor16sR insert and about 10ng of the digested vector (which had been cut with Bgl2) were ligated at11° C. overnight. One microliter of ligation reaction was electroporatedinto DH10B competent cells (Gibco BRL, Rockville, Md.) according tomanufacturer's direction and plated onto LB plates containing 50 mg/mlampicillin, and incubated overnight. Colonies were screened byrestriction analysis of DNA, which was prepared from 2 ml liquidcultures of individual colonies. The insert sequence of positive cloneswas verified by sequence analysis. A large-scale plasmid preparation wasdone using a Qiagen® Mega prep kit (Qiagen) according to manufacturer'sinstruction.

Similar methods are used to prepare non-zcytor16 subunits ofheterodimeric and multimeric receptors, such as CRF2-4 and IL-10R taggedwith Fc4.

C. Construction of Zcytor16 Mammalian Expression Vector ContainingZcytor16CEE, Zcytor16CFLG and Zcytor16CHIS

An expression vector is prepared for the expression of the soluble,extracellular domain of the zcytor16 polypeptide (e.g., amino acids22-231 of SEQ ID NO:2; SEQ ID NO:13), pC4zcytor16CEE, wherein theconstruct is designed to express a zcytor16 polypeptide comprised of thepredicted initiating methionine and truncated adjacent to the predictedtransmembrane domain, and with a C-terminal Glu-Glu tag (SEQ ID NO:10).

A zcytor16 DNA fragment comprising the zcytor16 extracellular cytokinebinding domain (e.g., SEQ ID NO:13) is created using PCR, and purified.The excised DNA is subcloned into a plasmid expression vector that has asignal peptide, e.g., the native zcytor16 signal peptide, tPA leader,and attaches a Glu-Glu tag (SEQ ID NO:10) to the C-terminus of thezcytor16 polypeptide-encoding polynucleotide sequence. Such anexpression vector mammalian expression vector contains an expressioncassette having a mammalian promoter, multiple restriction sites forinsertion of coding sequences, a stop codon and a mammalian terminator.The plasmid can also have an E. coli origin of replication, a mammalianselectable marker expression unit having an SV40 promoter, enhancer andorigin of replication, a DHFR gene and the SV40 terminator.

Restriction digested zcytor16 insert and previously digested vector areligated using standard molecular biological techniques, andelectroporated into competent cells such as DH10B competent cells (GIBCOBRL, Gaithersburg, Md.) according to manufacturer's direction and platedonto LB plates containing 50 mg/ml ampicillin, and incubated overnight.Colonies are screened by restriction analysis of DNA prepared fromindividual colonies. The insert sequence of positive clones is verifiedby sequence analysis. A large-scale plasmid preparation is done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

The same process is used to prepare the zcytor16 soluble homodimeric,heterodimeric or multimeric receptors (including non-zcytor16 solublereceptor subunits, such as, soluble CRF2-4 or IL-10R) with a C-terminalHIS tag, composed of 6 His residues in a row (SEQ ID NO:12); and aC-terminal FLAG (SEQ ID NO:11) tag, zcytor16CFLAG. To construct theseconstructs, the aforementioned vector has either the HIS or the FLAG®tag in place of the glu-glu tag (SEQ ID NO:10).

Example 2 Transfection And Expression of Soluble Receptor Polypeptides

The day before the transfection, BHK 570 cells (ATCC No. CRL-10314;ATCC, Manasas, Va.) were plated in a 10-cm plate with 50% confluence innormal BHK DMEM (Gibco/BRL High Glucose) media. The day of thetransfection, the cells were washed once with Serum Free (SF) DMEM,followed by transfection with the zcytor16sR/Fc4/pzmp20 expressionplasmids. Sixteen micrograms of zcytor16sR-Fc4 DNA construct (Example1B) were diluted into a total final volume of 640 μl SF DMEM. A dilutedLipofectAMINE™ (Gibco BRL, Gaithersburg, Md.) mixture (35 μlLipofectAMINE™ in 605 μL SF media) was added to the DNA mix, andincubated for 30 minutes at room temperature. Five milliliters of SFmedia was added to the DNA/LipofectAMINE™ mixture, which was then addedto BHK cells. The cells were incubated at 37° C./5% CO₂ for 5 hours,after which 6.4 ml of BHK media with 10% FBS was added. The cells wereincubated overnight at 37° C./5% CO₂.

Approximately 24 hours post-transfection, the BHK cells were split intoselection media with 1 μM methotrexate (MTX). The cells were repeatedlysplit in this manner until stable zcytor16sR-Fc4/BHK cell lines wereidentified. To detect the expression level of the zcytor16 solublereceptor fusion proteins, the transfected BHK cells were washed with PBSand incubated in SF media for 72 hours. The SF condition media wascollected and 20 μl of the sample was run on 10% SDS-PAGE gel underreduced conditions. The protein bands were transferred to nitrocellulosefilter by Western blot, and the fusion proteins were detected usinggoat-anti-human IgG/HRP conjugate (Jackson ImmunoResearch Laboratories,Inc, West Grove, Pa.). An expression vector containing a differentsoluble receptor fused to the Fc4 was used as a control. The expressionlevel of the stable zcytor16sR-Fc4/BHK cells was approximately 2 mg/L.

For protein purification, the transfected BHK cells were transferredinto T-162 flasks. Once the cells reached about 80% confluence, theywere washed with PBS and incubated in 100 ml SF media for 72 hours, andthen the condition media was collected for protein purification (Example11).

Example 3 Expression of Zcytor16 Soluble Receptor in E. coli

A. Construction of Expression Vector pCZR225 that Expresseshuzcytor16/MBP-6H Fusion Polypeptide

An expression plasmid containing a polynucleotide encoding a zcytor16soluble receptor fused C-terminally to maltose binding protein (MBP) isconstructed via homologous recombination. The fusion polypeptidecontains an N-terminal approximately 388 amino acid MBP portion fused tothe zcytor16 soluble receptor (e.g., SEQ ID NO:13). A fragment ofzcytor16 cDNA (SEQ ID NO:1) is isolated using PCR as described herein.Two primers are used in the production of the zcytor 16 fragment in astandard PCR reaction: (1) one containing about 40 bp of the vectorflanking sequence and about 25 bp corresponding to the amino terminus ofthe zcytor16, and (2) another containing about 40 bp of the 3′ endcorresponding to the flanking vector sequence and about 25 bpcorresponding to the carboxyl terminus of the zcytor16. Two μl of the100 μl PCR reaction is run on a 1.0% agarose gel with 1×TBE buffer foranalysis, and the expected approximately fragment is seen. The remainingPCR reaction is combined with the second PCR tube and precipitated with400 μl of absolute ethanol. The precipitated DNA used for recombininginto an appropriately restriction digested recipient vector pTAP98 toproduce the construct encoding the MBP-zcytor16 fusion, as describedbelow.

Plasmid pTAP98 is derived from the plasmids pRS316 and pMAL-c2. Theplasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P.and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E. coliexpression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP98 is constructed usingyeast homologous recombination. 100 ng of EcoRI cut pMAL-c2 isrecombined with 1 g Pvul cut pRS316, 1 μg linker, and 1 μg ScaI/EcoRIcut pRS316 are combined in a PCR reaction. PCR products are concentratedvia 100% ethanol precipitation.

Competent yeast cells (S. cerevisiae) are combined with about 10 μl of amixture containing approximately 1 μg of the zcytor16 receptor PCRproduct above, and 100 ng of digested pTAP98 vector, and electroporatedusing standard methods and plated onto URA-D plates and incubated at 30°C.

After about 48 hours, the Ura+ yeast transformants from a single plateare picked, DNA isolated, and transformed into electrocompetent E. colicells (e.g., MC1061, Casadaban et. al. J. Mol. Biol. 138, 179-207), andplated on MM/CA+AMP 100 mg/L plates (Pryor and Leiting, ProteinExpression and Purification 10:309-319, 1997). using standardprocedures. Cells are grown in MM/CA with 100 μg/ml Ampicillin for twohours, shaking, at 37° C. 1 ml of the culture is induced with 1 mM IPTG.2-4 hours later the 250 μl of each culture is mixed with 250 μl acidwashed glass beads and 250 μl Thomer buffer with 5% βME and dye (8Murea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples arevortexed for one minute and heated to 65° C. for 10 minutes. 20 μl areloaded per lane on a 4%-12% PAGE gel (NOVEX). Gels are run in 1×MESbuffer. The positive clones are designated pCZR225 and subjected tosequence analysis.

One microliter of sequencing DNA is used to transform strain BL21. Thecells are electropulsed at 2.0 kV, 25 μF and 400 ohms. Followingelectroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin. Cells are grownin MM/CA and induced with ITPG as described above. The positive clonesare used to grow up for protein purification of the huzcytor 16/MBP-6Hfusion protein using standard techniques.

Example 4 Zcytor16 Soluble Receptor Polyclonal Antibodies

Polyclonal antibodies are prepared by immunizing female New Zealandwhite rabbits with the purified huzcytor16/MBP-6H polypeptide (Example3), or the purified recombinant zcytor16CEE soluble receptor (Example 1;Example 11). The rabbits are each given an initial intraperitoneal (IP)injection of about 200 mg of purified protein in Complete Freund'sAdjuvant (Pierce, Rockford, Ill.) followed by booster IP injections of100 mg purified protein in Incomplete Freund's Adjuvant every threeweeks. Seven to ten days after the administration of the third boosterinjection, the animals are bled and the serum is collected. The rabbitsare then boosted and bled every three weeks.

The zcytor 16-specific polyclonal antibodies are affinity purified fromthe rabbit serum using an CNBr-SEPHAROSE ® 4B protein column (PharmaciaLKB) that is prepared using about 10 mg of the appropriate purifiedzcytor 16 polypeptide per gram CNBr-SEPHAROSE®, followed by 20× dialysisin PBS overnight. Zcytor 16-specific antibodies are characterized by anELISA titer check using 1 mg/ml of the appropriate protein antigen as anantibody target. The lower limit of detection (LLD) of the rabbitanti-zcytor 6 affinity purified antibodies is determined using standardmethods.

Example 5 Zcytor16 Receptor Monoclonal Antibodies

Zcytor16 receptor Monoclonal antibodies are prepared by immunizing maleBalbC mice (Harlan Sprague Dawley, Indianapolis, Ind.) with the purifiedrecombinant zcytor16 proteins described herein. The mice are each givenan initial intraperitoneal (IP) injection of 20 mg of purified proteinin Complete Freund's Adjuvant (Pierce, Rockford, Ill.) followed bybooster IP injections of 10 mg purified protein in Incomplete Freund'sAdjuvant every two weeks. Seven to ten days after the administration ofthe third booster injection, the animals are bled and the serum iscollected, and antibody titer assessed.

Splenocytes are harvested from high-titer mice and fused to murine SP2/0myeloma cells using PEG 1500 (Boerhinger Mannheim, UK) in two separatefusion procedures using a 4:1 fusion ratio of splenocytes to myelomacells (Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ColdSpring Harbor Press). Following 10 days growth post-fusion, specificantibody-producing hybridomas are identified by ELISA using purifiedrecombinant zcytor16 soluble receptor protein (Example 6C) as anantibody target and by FACS using Baf3 cells expressing the zcytor16sequence (Example 8) as an antibody target. The resulting hybridomaspositive by both methods are cloned three times by limiting dilution.

Example 6 Assessing Zcvtor16 Receptor Heterodimerization Using ORIGENAssay

Soluble zcytor16 receptor (Example 11), or gp130 (Hibi, M. et al., Cell63:1149-1157, 1990) are biotinylated by reaction with a five-fold molarexcess of sulfo-NHS-LC-Biotin (Pierce, Inc., Rockford, Ill.) accordingto the manufacturer's protocol. Soluble zcytor16 receptor and anothersoluble receptor subunit, for example, soluble IL-10R (sIL-10R) orCRF2-4 receptor (CRF2-4), soluble zcytor11 receptor (U.S. Pat. No.5,965,704) or soluble zcytor7 receptor (U.S. Pat. No. 5,945,511) arelabeled with a five fold molar excess of Ru-BPY-NHS (Igen, Inc.,Gaithersburg, Md.) according to manufacturer's protocol. Thebiotinylated and Ru-BPY-NHS-labeled forms of the soluble zcytor16receptor can be respectively designated Bio-zcytor16 receptor andRu-zcytor16; the biotinylated and Ru-BPY-NHS-labeled forms of the othersoluble receptor subunit can be similarly designated. Assays can becarried out using conditioned media from cells expressing a ligand, suchas IL-TIF, that binds zcytor16 heterodimeric receptors, or usingpurified IL-TIF.

For initial receptor binding characterization a panel of cytokines orconditioned medium are tested to determine whether they can mediatehomodimerization of zcytor16 receptor and if they can mediate theheterodimerization of zcytor16 receptor with the soluble receptorsubunits described above. To do this, 50 μl of conditioned media orTBS-B containing purified cytokine, is combined with 50 μl of TBS-B (20mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2) containing e.g., 400 ng/ml ofRu-zcytor16 receptor and Bio-zcytor16, or 400 ng/ml of Ru-zcytor16receptor and e.g., Bio-gp130, or 400 ng/ml of e.g., Ru-CRF2-4 andBio-zcytor16. Following incubation for one hour at room temperature, 30μg of streptavidin coated, 2.8 mm magnetic beads (Dynal, Inc., Oslo,Norway) are added and the reaction incubated an additional hour at roomtemperature. 200 μl ORIGEN assay buffer (Igen, Inc., Gaithersburg, Md.)is then added and the extent of receptor association measured using anM8 ORIGEN analyzer (Igen, Inc.).

Example 7 Construct for Generating a Zcytor16 Receptor Heterodimer

A vector expressing a secreted human zcytor16 heterodimer isconstructed. In this construct, the extracellular cytokine-bindingdomain of zcytor16 (e.g., SEQ ID NO:13) is fused to the heavy chain ofIgG gamma 1 (IgGγ1) while the extracellular portion of the heteromericcytokine receptor subunit (e.g., an CRF2-4, IL-9, IL-10, zcytor7,zcytor11, IL-4 receptor component) is fused to a human kappa light chain(human κ light chain).

A. Construction of IgG Gamma 1 and Human κ Light Chain Fusion Vectors

The heavy chain of IgGγ1 can be cloned into the Zem229R mammalianexpression vector (ATCC deposit No. 69447) such that any desiredcytokine receptor extracellular domain having a 5′ EcoRI and 3′ NheIsite can be cloned in resulting in an N-terminal extracellulardomain-C-terminal IgGγ1 fusion. The IgGγ1 fragment used in thisconstruct is made by using PCR to isolate the IgGγ1 sequence from aClontech hFetal Liver cDNA library as a template. PCR products arepurified using methods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with oligoswhich comprise an desired restriction site linker, into Zem229Rpreviously digested with and EcoRI using standard molecular biologytechniques disclosed herein.

The human κ light chain can be cloned in the Zem228R mammalianexpression vector (ATCC deposit No. 69446) such that any desiredcytokine receptor extracellular domain having a 5′ EcoRI site and a 3′KpnI site can be cloned in resulting in a N-terminal cytokineextracellular domain-C-terminal human κ light chain fusion. As a KpnIsite is located within the human κ light chain sequence, a specialprimer is designed to clone the 3′ end of the desired extracellulardomain of a cytokine receptor into this KpnI site: The primer isdesigned so that the resulting PCR product contains the desired cytokinereceptor extracellular domain with a segment of the human κ light chainup to the KpnI site. This primer preferably comprises a portion of atleast 10 nucleotides of the 3′ end of the desired cytokine receptorextracellular domain fused in frame 5′ to fragment cleaved at the KpnIsite. The human κ light chain fragment used in this construct is made byusing PCR to isolate the human κ light chain sequence from the sameClontech human Fetal Liver cDNA library used above. PCR products arepurified using methods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with theMluI/EcoRI linker described above, into Zem228R previously digested withand EcoRI using standard molecular biology techniques disclosed herein.

B. Insertion of Zcytor16 Receptor or Heterodimeric Subunit ExtracellularDomains into Fusion Vector Constructs

Using the construction vectors above, a construct having zcytor16 fusedto IgGγ1 is made. This construction is done by PCRing the extracellularcytokine-binding domain of zcytor16 receptor (SEQ ID NO:13) from atonsil cDNA library (Clontech) or plasmid (Example 1A) using standardmethods and oligos that provide EcoRI and NheI restriction sites. Theresulting PCR product is digested with EcoRI and NheI, gel purified, asdescribed herein, and ligated into a previously EcoRI and NheI digestedand band-purified Zem229R/IgGγ1 described above. The resulting vector issequenced to confirm that the zcytor16/IgG gamma 1 fusion is correct.

A separate construct having a heterodimeric cytokine receptor subunitextracellular domain fused to κ light is also constructed as above. TheCRF2-4/human κ light chain construction is performed as above by PCRingfrom, e.g., a lymphocyte cDNA library (Clontech) using standard methods,and oligos that provide EcoRI and KpnI restriction sites. The resultingPCR product is digested with EcoRI and KpnI and then ligating thisproduct into a previously EcoRI and KpnI digested and band-purifiedZem228R/human κ light chain vector described above. The resulting vectoris sequenced to confirm that the cytokine receptor subunit/human κ lightchain fusion is correct.

D. Co-Expression of the Zcytor16 and Heterodimeric Cytokine ReceptorSubunit Extracellular Domain

Approximately 15 μg of each of vectors above, are co-transfected intomammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) usingLipofectaminePlus™ reagent (Gibco/BRL), as per manufacturer'sinstructions. The transfected cells are selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis,Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resulting pool oftransfectants is selected again in 10 μm of MTX and 0.5 mg/ml G418 forabout 10 days.

The resulting pool of doubly selected cells is used to generate protein.Three Factories (Nunc, Denmark) of this pool are used to generate 10 Lof serum free conditioned medium. This conditioned media is passed overa 1 ml protein-A column and eluted in about 10, 750 microliterfractions. The fractions having the highest protein concentration arepooled and dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzedmaterial is submitted for amino acid analysis (AAA) using routinemethods.

Example 8 Determination of Receptor Subunits that Heterodimerize orMultimerize with Zcytor16 Receptor Using a Proliferation Assay

Using standard methods described herein, cells expressing aBaF3/zcytor16-MPL chimera (wherein the extracellular domain of thezcytor16 (e.g., SEQ ID NO:13) is fused in frame to the intracellularsignaling domain of the mpl receptor) are tested for proliferativeresponse in the presence of IL-TIF. Such cells serve as a bioassay cellline to measure ligand binding of monomeric or homodimeric zcytor16receptors. In addition, BaF3/zcytor16-MPL chimera cells transfected withan additional heterodimeric cytokine receptor subunit can be assessedfor proliferative response in the presence of IL-TIF. In the presence ofIL-TIF, if the BaF3/zcytor16-MPL cells signal, this would suggest thatzcytor16 receptor can homodimerize to signal. Transfection of theBaF3/MPL-zcytor16 cell line with and additional MPL-class II cytokinereceptor fusion that signals in the presence of the IL-TIF ligand, suchas CRF2-4, determines which heterodimeric cytokine receptor subunits arerequired for zcytor16 receptor signaling. Use of MPL-receptor fusionsfor this purpose alleviates the requirement for the presence of anintracellular signaling domain for the zcytor16 receptor.

Each independent receptor complex cell line is then assayed in thepresence of IL-TIF and proliferation measured using routine methods(e.g., Alamar Blue assay). The BaF3/MPL-zcytor16 bioassay cell lineserves as a control for the monomeric or homodimeric receptor activity,and is thus used as a baseline to compare signaling by the variousreceptor complex combinations. The untransfected bioassay cell lineserves as a control for the background activity, and is thus used as abaseline to compare signaling by the various receptor complexcombinations. A BaF3/MPL-zcytor16 without ligand (IL-TIF) is also usedas a control. The IL-TIF in the presence of the correct receptorcomplex, is expected to increase proliferation of the BaF3/zcytor16-MPLreceptor cell line approximately 5 fold over background or greater inthe presence of IL-TIF. Cells expressing the components of zcytor16heterodimeric and multimeric receptors should proliferate in thepresence of IL-TIF.

Example 9 Reconstitution of Zcytor16 Receptor In Vitro

To identify components involved in the zcytor16-signaling complex,receptor reconstitution studies are performed as follows. BHK 570 cells(ATCC No. CRL-10314) transfected, using standard methods describedherein, with a luciferase reporter mammalian expression vector plasmidserve as a bioassay cell line to measure signal transduction responsefrom a transfected zcytor16 receptor complex to the luciferase reporterin the presence of IL-TIF. BHK cells do not endogenously express thezcytor16 receptor. An exemplary luciferase reporter mammalian expressionvector is the KZ134 plasmid which was constructed with complementaryoligonucleotides that contain STAT transcription factor binding elementsfrom 4 genes. A modified c-fos S is inducible element (m67SIE, or hSIE)(Sadowski, H. et al., Science 261:1739-1744, 1993), the p21 SIE1 fromthe p21 WAF1 gene (Chin, Y. et al., Science 272:719-722, 1996), themammary gland response element of the β-casein gene (Schmitt-Ney, M. etal., Mol. Cell. Biol. 11:3745-3755, 1991), and a STAT inducible elementof the Fcg RI gene, (Seidel, H. et al., Proc. Natl. Acad. Sci.92:3041-3045, 1995). These oligonucleotides contain Asp718-XhoIcompatible ends and were ligated, using standard methods, into arecipient firefly luciferase reporter vector with a c-fos promoter(Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998) digestedwith the same enzymes and containing a neomycin selectable marker. TheKZ134 plasmid is used to stably transfect BHK, or BaF3 cells, usingstandard transfection and selection methods, to make a BHK/KZ134 orBaF3/KZ134 cell line respectively.

The bioassay cell line is transfected with zcytor16-mpl fusion receptoralone, or co-transfected along with one of a variety of other knownreceptor subunits. Receptor complexes include but are not limited tozcytor16-mpl receptor only, various combinations of zcytor16-mplreceptor with one or more of the CRF2-4, IL-9, IL-10, zcytor11, zcytor7class II cytokine receptor subunits, or IL-4 receptor components, or theIL-2 receptor components (IL-2Rα, IL-2Rβ, IL-2Rγ); zcytor16-mpl receptorwith one or more of the IL-4/IL-13 receptor family receptor components(IL-4Rα, IL-13Rα, IL-13Rα′), as well as other Interleukin receptors(e.g., IL-15 Rα, IL-7Rα, IL-9Rα, IL-21R (zalpha11; WIPO publication WOOO/17235; Parrish-Novak, J et al., Nature 408:57-63, 2000)). Eachindependent receptor complex cell line is then assayed in the presenceof cytokine-conditioned media or purified cytokines and luciferaseactivity measured using routine methods. The untransfected bioassay cellline serves as a control for the background luciferase activity, and isthus used as a baseline to compare signaling by the various receptorcomplex combinations. The conditioned medium or cytokine that binds thezyctor16 receptor in the presence of the correct receptor complex, isexpected to give a luciferase readout of approximately 5 fold overbackground or greater.

As an alternative, a similar assay can be performed whereinBaf3/zcytor16-mpl cell lines are co-transfected as described above andproliferation measured (Example 8).

Example 10 COS Cell Transfection and Secretion Trap

COS cell transfections were performed as follows: A mixture of 0.5 μgDNA and 5 μl lipofectamine (Gibco BRL) in 92 ul serum free DMEM media(55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mginsulin, 1 μg selenium and 5 mg fetuin in 500 ml DMEM) was incubated atroom temperature for 30 minutes and then 400 μl serum free DMEM mediaadded. A 500 μl mixture was added onto COS cells plated on 12-welltissue culture plate at 1.5×10⁵ COS cells/well and previously incubatedfor 5 hours at 37° C. An additional 500 μl 20% FBS DMEM media (100 mlFBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) wasadded and the plates were incubated overnight.

The secretion trap was performed as follows: Media was rinsed off cellswith PBS and fixed for 15 minutes with 1.8% Formaldehyde in PBS. Cellswere then washed with TNT (0.1M Tris-HCl, 0.15M NaCl, and 0.05% Tween-20in H₂O). Cells were permeated with 0.1% Triton-X in PBS for 15 minutesand washed again with TNT. C ells were blocked for 1 hour with TNB (0.1MTris-HCl, 0.15M NaCl and 0.5% Blocking Reagent (NEN RenaissanceTSA-Direct Kit; NEN) in H₂O. Cells were again washed with TNT. Cellswere then incubated for 1 hour with 1-3 μg/ml zcytor16 soluble receptorFc4 fusion protein (zcytor16sR-Fc4) (Example 11) in TNB. Cells werewashed with TNT, and then incubated for another hour with 1:200 dilutedgoat-anti-human Ig-HRP (Fc specific; Jackson ImmunoResearchLaboratories, Inc.) in TNB. Cells were again washed with TNT. Antibodiespositively binding to the zcytor16sR-Fc4 were detected with fluoresceintyramide reagent diluted 1:50 in dilution buffer (NEN kit) and incubatedfor 4-6 minutes. Cells were again washed with TNT. Cells were preservedwith Vectashield Mounting Media (Vector Labs) diluted 1:5 in TNT. Cellswere visualized using FITC filter on fluorescent microscope.

Since zcytor16 is a Class II cytokine receptor, the binding ofzcytor16sR/Fc4 fusion protein with known or orphan Class II cytokineswas tested. The pZP7 expression vectors containing cDNAs of cytokines(including human IL-TIF, interferon alpha, interferon beta, interferongamma, IL-10, amongst others) were transfected into COS cells, and thebinding of zcytor16sR/Fc4 to transfected COS cells were carried outusing the secretion trap assay described above. Human IL-TIF showedpositive binding. Based on these data, human IL-TIF and zcytor16 is apotential ligand-receptor pair.

Example 11 Purification of Zcytor16-Fc4 Polypeptide From Transfected BHK570 Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zcytor16 polypeptidecontaining C-terminal fusion to human Fc4 (zcytor16-Fc4; Example 1).About 16,500 ml of conditioned media from BHK 570 cells transfected withzcytor16-Fc4 (Example 2) was filtered through a 0.2 um sterilizingfilter and then supplemented with a solution of protease inhibitors, tofinal concentrations of, 0.001 mM leupeptin (Boerhinger-Mannheim,Indianapolis, Ind.), 0.001 mM pepstatin (Boerhinger-Mannheim) and 0.4 mMPefabloc (Boerhinger-Mannheim). A Poros protein A50 column (20 ml bedvolume, Applied Biosystems) was packed and washed with 400 ml PBS(Gibco/BRL) The supplemented conditioned media was passed over thecolumn with a flow rate of 15 ml/minute, followed by washing with 800 mlPBS (BRL/Gibco). Zcytor16-Fc4 was eluted from the column with 0.1 MGlycine pH 3.0 and 5 ml fractions were collected directly into 0.5 ml 2MTris pH 7.8, to adjust the final pH to 7.4 in the fractions.

Column performance was characterized through western blotting ofreducing SDS-PAGE gels of the starting media and column pass through.Western blotting used anti-human IgG HRP (Amersham) antibody, whichshowed an immunoreactive protein at 60,000 Da in the starting media,with nothing in the pass through, suggesting complete capture. Theprotein A50 eluted fractions were characterized by reducing SDS PAGEgel. This gel showed an intensely Coomassie stained band at 60,000 Da infractions 3 to 11. Fractions 3 to 11 were pooled.

Protein A 50 elution pool was concentrated from 44 ml to 4 ml using a30,000 Da Ultrafree Biomax centrifugal concentrator (15 ml volume,Millipore). A Sephacryl S-300 gel filtration column (175 ml bed volume;Pharmacia) was washed with 350 ml PBS (BRL/Gibco). The concentrated poolwas injected over the column with a flow rate of 1.5 ml/min, followed bywashing with 225 ml PBS (BRL/Gibco). Eluted peaks were collected into 2ml fractions.

Eluted fractions were characterized by reducing and non-reducing silverstained (Geno Technology) SDS PAGE gels. Reducing silver stained SDSPAGE gels showed an intensely stained band at 60,000 Da in fractions14-31, while non-reducing silver stained SDS PAGE gels showed anintensely stained band at 160,000 Da in fractions 14-31. Fractions 1-13showed many bands of various sizes. Fractions 14-31 were pooled,concentrated to 22 ml using 30,000 Da Ultrafree Biomax centrifugalconcentrator (15 ml volume, Millipore). This concentrate was filteredthrough a 0.2 μm Acrodisc sterilizing filter (Pall Corporation).

The protein concentration of the concentrated pooled fractions wasperformed by BCA analysis (Pierce, Rockford, Ill.) and the material wasaliquoted, and stored at −80° C. according to our standard procedures.The concentration of the pooled fractions was 1.50 mg/ml.

Example 12 Human Zcytor16 Tissue Distribution in Tissue Panels UsingNorthern Blot and PCR

A. Human Zcytor16 Tissue Distribution using Northern Blot and Dot Blot

Northern blot analysis was performed using Human Multiple TissueNorthern Blots I, II, III (Clontech) and an in house generated U-937northern blot. U-937 is a human monoblastic promonocytic cell line. ThecDNA probe was generated using oligos ZC25,963 (SEQ ID NO:16) andZC28,354 (SEQ ID NO:17). The PCR conditions were as follows: 94° for 1minute; 30 cycles of 94°, 15 seconds; 60°, 30 seconds; 72°, 30 secondsand a final extension for 5 minutes at 72°. The 364 bp product was gelpurified by gel electrophoresis on a 1% TBE gel and the band was excisedwith a razor blade. The cDNA was extracted from the agarose using theQIAquick Gel Extraction Kit (Qiagen). 94 ng of this fragment wasradioactively labeled with ³²P-dCTP using Rediprime II (Amersham), arandom prime labeling system, according to the manufacturer'sspecifications. Unincorporated radioactivity was removed using aNuc-Trap column (Stratagene) according to manufacturer's instructions.Blots were prehybridized at 65° for 3 hours in ExpressHyb (Clontech)solution. Blots were hybridized overnight at 65° in Expresshyb solutioncontaining 1.0×10⁶ cpm/ml of labeled probe, 0.1 mg/ml of salmon spermDNA and 0.5 μg/ml of human cot-1 DNA. Blots were washed in 2×SSC, 0.1%SDS at room temperature with several solution changes then washed in0.1×SSC. 0.1% SDS at 55° for 30 minutes twice. Transcripts ofapproximately 1.6 kb and 3.0 kb size were detected in spleen andplacenta, but not other tissues examined. The same sized transcriptsplus an additional approximate 1.2 kb transcript was detected in U-937cell line.

B. Tissue Distribution in Tissue cDNA Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor16 expressionusing PCR. The panel was made in-house and contained 94 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines are shown in Table 5, below. The cDNAs came from in-houselibraries or marathon cDNAs from in-house RNA preps, Clontech RNA, orInvitrogen RNA. The marathon cDNAs were made using the marathon-Ready™kit (Clontech, Palo Alto, Calif.) and QC tested with clathrin primersZC21195 (SEQ ID NO:18) and ZC21196 (SEQ ID NO:19) and then diluted basedon the intensity of the clathrin band. To assure quality of the panelsamples, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo ZC14,063 (SEQ ID NO:20) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:21) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:22); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a human genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. The PCR reactions wereset up using oligos ZC25,963 (SEQ ID NO:16) and ZC27,659 (SEQ ID NO:23),Advantage 2 DNA Polymerase Mix (Clontech) and Rediload dye (ResearchGenetics, Inc., Huntsville, Ala.). The amplification was carried out asfollow: 1 cycle at 94° C. for 2 minutes, 30 cycles of 94° C. for 20seconds, 58° C. for 30 seconds and 72° C. for 1 minute, followed by 1cycle at 72° C. for 5 minutes. About 10 μl of the PCR reaction productwas subjected to standard Agarose gel electrophoresis using a 2% agarosegel. The correct predicted DNA fragment size was not observed in anytissue or cell line. Subsequent experiments showing expression ofzcytor16 indicated that the negative results from this panel were likelydue to the primers used.

TABLE 5 # # Tissue/Cell line samples Tissue/Cell line samples Adrenalgland 1 Bone marrow 3 Bladder 1 Fetal brain 3 Bone Marrow 1 Islet 2Brain 1 Prostate 3 Cervix 1 RPMI #1788 (ATCC # CCL-156) 2 Colon 1 Testis4 Fetal brain 1 Thyroid 2 Fetal heart 1 WI38 (ATCC # CCL-75 2 Fetalkidney 1 ARIP (ATCC # CRL-1674 - rat) 1 Fetal liver 1 HaCat - humankeratinocytes 1 Fetal lung 1 HPV (ATCC # CRL-2221) 1 Fetal muscle 1Adrenal gland 1 Fetal skin 1 Prostate SM 2 Heart 2 CD3+ selected PBMC's1 Ionomycin + PMA stimulated K562 (ATCC # 1 HPVS (ATCC # CRL-2221) - 1CCL-243) selected Kidney 1 Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2Lymph node 1 Salivary gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3Pancreas 1 Platelet 1 Pituitary 1 HBL-100 1 Placenta 1 Renal mesangial 1Prostate 1 T-cell 1 Rectum 1 Neutrophil 1 Salivary Gland 1 MPC 1Skeletal muscle 1 Hut-102 (ATCC # TIB-162) 1 Small intestine 1Endothelial 1 Spinal cord 1 HepG2 (ATCC # HB-8065) 1 Spleen 1 Fibroblast1 Stomach 1 E. Histo 1 Testis 2 Thymus 1 Thyroid 1 Trachea 1 Uterus 1Esophagus tumor 1 Gastric tumor 1 Kidney tumor 1 Liver tumor 1 Lungtumor 1 Ovarian tumor 1 Rectal tumor 1 Uterus tumor 1

An additional panel of cDNAs from human tissues was screened forzcytor16 expression using PCR. This panel was made in-house andcontained 77 marathon cDNA and cDNA samples from various normal andcancerous human tissues and cell lines are shown in Table 6, below.Aside from the PCR reaction, the assay was carried out as per above. ThePCR reactions were set up using oligos ZC25,963 (SEQ ID NO:30) andZC25,964 (SEQ ID NO:31), Advantage 2 DNA Polymerase Mix (Clontech) andRediload dye (Research Genetics, Inc., Huntsville, Ala.). Theamplification was carried out as follow: 1 cycle at 94° C. for 1 minute,38 cycles of 94° C. for 10 seconds, 60° C. for 30 seconds and 72° C. for30 seconds, followed by 1 cycle at 72° C. for 5 minutes. The correctpredicted DNA fragment size was observed in bone marrow, fetal heart,fetal kidney, fetal muscle, fetal skin, heart, mammary gland, placenta,salivary gland, skeletal muscle, small intestine, spinal cord, spleen,kidney, fetal brain, esophageal tumor, uterine tumor, stomach tumor,ovarian tumor, rectal tumor, lung tumor and RPMI-1788 (a B-lymphocytecell line). Zcytor16 expression was not observed in the other tissuesand cell lines tested in this panel. The expression pattern of zcytor16shows expression in certain tissue-specific tumors especially, e.g.,ovarian cancer, stomach cancer, uterine cancer, rectal cancer, lungcancer and esophageal cancer, where zcytor 16 is not expressed in normaltissue, but is expressed in the tumor tissue. One of skill in the artwould recognize that the polynucleotides, polypeptides, antibodies, andbinding partners of the present invention can be used as a diagnostic todetect cancer, or cancer tissue in a biopsy, tissue, or histologicsample, particularly e.g., ovarian cancer, stomach cancer, uterinecancer, rectal cancer, lung cancer and esophageal cancer tissue. Suchdiagnostic uses for the molecules of the present invention are known inthe art and described herein.

In addition, because the expression pattern of zcytor16, one of IL-TIF'sreceptors, shows expression in certain specific tissues as well astissue-specific tumors, binding partners including the natural liganed,IL-TIF, can also be used as a diagnostic to detect specific tissues(normal or abnormal), cancer, or cancer tissue in a biopsy, tissue, orhistologic sample, where IL-TIF receptors are expressed, andparticularly e.g., ovarian cancer, stomach cancer, uterine cancer,rectal cancer, lung cancer and esophageal cancer tissue. IL-TIF can alsobe used to target other tissues wherein its receptors, e.g., zcytor16and zcytor11 are expressed. Moreover, such binding partners could beconjugated to chemotherapeutic agents, toxic moieties and the like totarget therapy to the site of a tumor or diseased tissue. Suchdiagnostic and targeted therapy uses are known in the art and describedherein.

A commercial 1st strand cDNA panel (Human Blood Fractions MTC Panel,Clontech, Palo Alto, Calif.) was also assayed as above. The panelcontained the following samples: mononuclear cells, activatedmononuclear cells, resting CD4+ cells, activated CD4+ cells, restingCD8+ cells, activated CD8+ cells, resting CD14+ cells, resting CD19+cells and activated CD19+ cells. Activated CD4+ cells and activatedCD19+ cells showed zcytor16 expression, whereas the other cells tested,including resting CD4+ cells and resting CD19+ cells, did not.

TABLE 6 Tissue # samples Tissue # samples adrenal gland 1 bladder 1 bonemarrow 3 brain 2 cervix 1 colon 1 fetal brain 3 fetal heart 2 fetalkidney 1 fetal liver 2 fetal lung 1 fetal skin 1 heart 2 fetal muscle 1kidney 2 liver 1 lung 1 lymph node 1 mammary gland 1 melanoma 1 ovary 1pancreas 1 pituitary 2 placenta 3 prostate 3 rectum 1 salivary gland 2skeletal muscle 1 small intestine 1 spinal cord 2 spleen 1 uterus 1stomach 1 adipocyte library 1 testis 5 islet 1 thymus 1 prostate SMC 1thyroid 2 RPMI 1788 1 trachea 1 W138 1 esophageal tumor 1 lung tumor 1liver tumor 1 ovarian tumor 1 rectal tumor 1 stomach tumor 1 uterinetumor 2 CD3+ library 1 HaCAT library 1 HPV library 1 HPVS library 1 MG63library 1 K562 1C. Tissue Distribution in Human Tissue and Cell Line RNA Panels UsingRT-PCR

A panel of RNAs from human cell lines was screened for zcytor16expression using RT-PCR. The panels were made in house and contained 84RNAs from various normal and cancerous human tissues and cell lines asshown in Tables 7-10 below. The RNAs were made from in house orpurchased tissues and cell lines using the RNAeasy Midi or Mini Kit(Qiagen, Valencia, Calif.). The panel was set up in a 96-well formatwith 100 ngs of RNA per sample. The RT-PCR reactions were set up usingoligos ZC25,963 (SEQ ID NO:30) and ZC25,964 (SEQ ID NO:31), Rediload dyeand SUPERSCRIPT One Step RT-PCR System (Life Technologies, Gaithersburg,Md.). The amplification was carried out as follows: one cycle at 55° for30 minutes followed by 40 cycles of 94°, 15 seconds; 59°, 30 seconds;72°, 30 seconds; then ended with a final extension at 72° for 5 minutes.8 to 10 μls of the PCR reaction product was subjected to standardAgarose gel electrophoresis using a 4% agarose gel. The correctpredicted cDNA fragment size of 184 bps was observed in cell linesU-937, HL-60, ARPE-19, HaCat#1, HaCat#2, HaCat#3, and HaCat#4; bladder,cancerous breast, normal breast adjacent to a cancer, bronchus, colon,ulcerative colitis colon, duodenum, endometrium, esophagus,gastro-esophageal, heart left ventricle, heart ventricle, ileum, kidney,lung, lymph node, lymphoma, mammary adenoma, mammary gland, cancerousovary, pancreas, parotid and skin, spleen lymphoma and small bowel.Zcytor16 expression was not observed in the other tissues and cell linestested in this panel.

Zcytor16 is detectably expressed by PCR in normal tissues: such as, thedigestive system, e.g., esophagus, gastro-esophageal, pancreas,duodenum, lleum, colon, small bowel; the female reproductive system,e.g., mammary gland, endometrium, breast (adjacent to canceroustissues); and others systems, e.g., lymph nodes, skin, parotid, bladder,bronchus, heart ventricles, and kidney. Moreover, Zcytor16 is detectablyexpressed by PCR in several human tumors: such as tumors associated withfemale reproductive tissues e.g., mammary adenoma, ovary cancer, uterinecancer, other breast cancers; and other tissues such as lymphoma,stomach tumor, and lung tumor. The expression of zcytor16 is found innormal tissues of female reproductive organs, and in some tumorsassociated with these organs. As such, zcytor16 can serve as a markerfor these tumors wherein the zcytor16 may be over-expressed. Severalcancers positive for zcytor16 are associated with ectodermal/epithelialorigin (mammary adenoma, and other breast cancers). Hence, zcytor16 canserve as a marker for epithelial tissue, such as epithelial tissues inthe digestive system and female reproductive organs (e.g., endometrialtissue, columnar epithelium), as well as cancers involving epithelialtissues. Moreover, in a preferred embodiment, zcytor16 can serve as amarker for certain tissue-specific tumors especially, e.g., ovariancancer, stomach cancer, uterine cancer, rectal cancer, lung cancer andesophageal cancer, where zcytor 16 is not expressed in normal tissue,but is expressed in the tumor tissue. Use of polynucleotides,polypeptides, and antibodies of the present invention for diagnosticpurposes are known in the art, and disclosed herein.

TABLE 7 Tissue # samples Tissue # samples adrenal gland 6 duodenum 1bladder 3 endometrium 5 brain 2 cancerous 1 endometrium brain meningioma1 gastric cancer 1 breast 1 esophagus 7 cancerous breast 4gastro-esophageal 1 normal breast adjacent to 5 heart aorta 1 cancerbronchus 3 heart left ventricle 4 colon 15 heart right ventricle 2cancerous colon 1 heart ventricle 1 normal colon adjacent to 1 ileum 3cancer ulcerative colitis colon 1 kidney 15 cancerous kidney 1

TABLE 8 Tissue/Cell Line # samples Tissue/Cell Line # samples 293 1HBL-100 1 C32 1 Hs-294T 1 HaCat#1 1 Molt4 1 HaCat#2 1 RPMI 1 HaCat#3 1U-937 1 HaCat#4 1 A-375 1 WI-38 1 HCT-15 1 WI-38 + 2 um ionomycin 1HT-29 1 #1 WI-38 + 2 um ionomycin 1 MRC-5 1 #2 WI-38 + 5 um ionomycin#11 RPT-1 1 WI-38 + 5 um ionomycin#2 1 RPT-2 1 Caco-2, 1 WM-115 1 Caco-2,differentiated 1 A-431 1 DLD-1 1 WERI-Rb-1 1 HRE 1 HEL-92.1.7 1 HRCE 1HuH-7 1 MCF7 1 MV-4-11 1 PC-3 1 U-138 1 TF-1 1 CCRF-CEM 1 5637 1 Y-79 1143B 1 A-549 1 ME-180 1 EL-4 1 prostate epithelia 1 HeLa 229 1 U-2 OS 1HUT 78 1 T-47D 1 NCI-H69 1 Mg-63 1 SaOS2 1 Raji 1 USMC 1 U-373 MG 1UASMC 2 A-172 1 AoSMC 1 CRL-1964 1 UtSMC 1 CRL-1964 + butryic acid 1HepG2 1 HUVEC 1 HepG2-IL6 1 SK-Hep-1 1 NHEK#1 1 SK-Lu-1 1 NHEK#2 1Sk-MEL-2 1 NHEK#3 1 K562 1 NHEK#4 1 BeWo 1 ARPE-19 1 FHS74.Int 1 G-361 1HL-60 1 HISM 1 Malme 3M 1 3AsubE 1 FHC 1 INT407 1 HREC 1

TABLE 9 Tissue # samples Tissue # samples liver 10 lung 13 lymph node 1cancerous lung 2 lymphoma 4 normal lung adjacent to 1 cancer mammaryadenoma 1 muscle 3 mammary gland 3 neuroblastoma 1 melinorioma 1 omentum2 osteogenic sarcoma 2 ovary 6 pancreas 4 cancerous ovary 2 skin 5parotid 7 sarcoma 2 salivary gland 4

TABLE 10 Tissue # samples Tissue # samples small bowel 10 uterus 11spleen 3 uterine cancer 1 spleen lymphoma 1 thyroid 9 stomach 13 stomachcancer 1

Example 13 Construction of Expression Vector Expressing Full-lengthZcytor11

The entire zcytor11 receptor (commonly owned U.S. Pat. No. 5,965,704)was isolated by digestion with EcoRI and XhoI from plasmid pZP7P,containing full-length zcytor11 receptor cDNA (SEQ ID NO:24) and apuromycin resistance gene. The digest was run on a 1% low melting pointagarose (Boerhinger Mannheim) gel and the approximately 1.5 kb zcytor11cDNA was isolated using Qiaquick™ gel extraction kit (Qiagen) as permanufacturer's instructions. The purified zcytor11 cDNA was insertedinto an expression vector as described below.

Recipient expression vector pZP7Z was digested with EcoRI (BRL) and XhoI(Boehringer Mannheim) as per manufacturer's instructions, and gelpurified as described above. This vector fragment was combined with theEcoRI and XhoI cleaved zcytor11 fragment isolated above in a ligationreaction. The ligation was run using T4 Ligase (BRL) at 12° C.overnight. A sample of the ligation was electroporated in to DH10BelectroMAX™ electrocompetent E. coli cells (25 μF, 200Ω, 1.8V).Transformants were plated on LB+Ampicillin plates, and single colonieswere picked into 2 ml LB+Ampicillin and grown overnight. Plasmid DNA wasisolated using Wizard Minipreps (Promega), and each was digested withEcoRI and XhoI to confirm the presence of insert. The insert wasapproximately 1.5 kb, and was full-length. Digestion with SpeI and PstIwas used to confirm the identity of the vector.

Example 14 Construction of BaF3 Cells Expressing the CRF2-4 Receptor(BaF3/CRF2-4 Cells) and BaF3 Cells Expressing the CRF2-4 Receptor withthe Zcytor11 Receptor (BaF3/CRF2-4/Zcytor11 Cells)

BaF3 cells expressing the full-length CFR2-4 receptor were constructed,using 30 μg of a CFR2-4 expression vector, described below. The BaF3cells expressing the CFR2-4 receptor were designated as BaF3/CFR2-4.These cells were used as a control, and were further transfected withfull-length zcytor11 receptor (U.S. Pat. No. 5,965,704) and used toconstruct a screen for IL-TIF activity as described below.

A. Construction of BaF3 Cells Expressing the CRF2-4 Receptor

The full-length cDNA sequence of CRF2-4 (Genbank Accession No. Z17227)was isolated from a Daudi cell line cDNA library, and then cloned intoan expression vector pZP7P, as described in Example 6.

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, CRF2-4/pZP7P was prepared andpurified using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer'sinstructions. For electroporation, BaF3 cells were washed once inserum-free RPMI media and then resuspended in serum-free RPMI media at acell density of 10⁷ cells/ml. One ml of resuspended BaF3 cells was mixedwith 30 μg of the CRF2-4/pZP7P plasmid DNA and transferred to separatedisposable electroporation chambers (GIBCO BRL). Following a 15-minuteincubation at room temperature the cells were given two serial shocks(800 IFad/300 V.; 1180 IFad/300 V.) delivered by an electroporationapparatus (CELL-PORATOR™; GIBCO BRL). After a 5-minute recovery time,the electroporated cells were transferred to 50 ml of complete media andplaced in an incubator for 15-24 hours (37° C., 5% CO₂). The cells werethen spun down and resuspended in 50 ml of complete media containing 2μg/ml puromycin in a T-162 flask to isolate the puromycin-resistantpool. Pools of the transfected BaF3 cells, hereinafter calledBaF3/CRF2-4 cells, were assayed for signaling capability as describedbelow. Moreover these cells were further transfected with zcytor11receptor as described below.

B. Construction of BaF3 Cells Expressing CRF2-4 and Zcytor11 Receptors

BaF3/CRF2-4 cells expressing the full-length zcytor11 receptor wereconstructed as per Example 5A above, using 30 μg of the zcytor11expression vector, described in Example 6 above. Following recovery,transfectants were selected using 200 μg/ml zeocin and 2 μg/mlpuromycin. The BaF3/CRF2-4 cells expressing the zcytor11 receptor weredesignated as BaF3/CRF2-4/zcytor11 cells. These cells were used toscreen for IL-TIF activity as well as zcytor16 antagonist activitydescribed IN Example 15.

Example 15 Screening for IL-TIF Antagonist Activity UsingBaF3/CRF2-4/Zcytor11 Cells Using an Alamar Blue Proliferation Assay

A. Screening for IL-TIF Activity Using BaF3/CRF2-4/zcytor11 Cells Usingan Alamar Blue Proliferation Assay

Purified IL-TIF-CEE (Example 19) was used to test for the presence ofproliferation activity as described below. Purified zcytor16-Fc4(Example 11) was used to antagonize the proliferative response of theIL-TIF in this assay as described below.

BaF3/CRF2-4/zcytor11 cells were spun down and washed in the completemedia, described in Example 7A above, but without mIL-3 (hereinafterreferred to as “mIL-3 free media”). The cells were spun and washed 3times to ensure the removal of the mIL-3. Cells were then counted in ahemacytometer. Cells were plated in a 96-well format at 5000 cells perwell in a volume of 100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/CRF2-4/zcytor11 cells was assessed usingIL-TIF-CEE protein diluted with mIL-3 free media to 50, 10, 2, 1, 0.5,0.25, 0.13, 0.06 ng/ml concentrations. 100 μl of the diluted protein wasadded to the BaF3/CRF2-4/zcytor11 cells. The total assay volume is 200μl. The assay plates were incubated at 37° C., 5% CO₂ for 3 days atwhich time Alamar Blue (Accumed, Chicago, Ill.) was added at 20 μl/well.Plates were again incubated at 37° C., 5% CO₂ for 24 hours. Alamar Bluegives a fluourometric readout based on number of live cells, and is thusa direct measurement of cell proliferation in comparison to a negativecontrol. Plates were again incubated at 37° C., 5% CO₂ for 24 hours.Plates were read on the Fmax™ plate reader (Molecular Devices Sunnyvale,Calif.) using the SoftMax™ Pro program, at wavelengths 544 (Excitation)and 590 (Emmission). Results confirmed the dose-dependent proliferativeresponse of the BaF3/CRF2-4/zcytor11 cells to IL-TIF-CEE. The response,as measured, was approximately 15-fold over background at the high endof 50 ng/ml down to a 2-fold induction at the low end of 0.06 ng/ml. TheBaF3 wild type cells, and BaF3/CRF2-4 cells did not proliferate inresponse to IL-TIF-CEE, showing that IL-TIF is specific for theCRF2-4/zcytor11 heterodimeric receptor.

In order to determine if zcytor16 is capable of antagonizing IL-TIFactivity, the assay described above was repeated using purified solublezcytor16/Fc4. When IL-TIF was combined with zcytor16 at 10 μg/ml, theresponse to IL-TIF at all concentrations was brought down to background.That the presence of soluble zcytor16 ablated the proliferative effectsof IL-TIF demonstrates that it is a potent antagonist of the IL-TIFligand.

Example 16 IL-TIF Activation of a Reporter Mini-Gene in MES 13 Cells andInhibition of Activity by Zcytor16-Fc4

MES 13 cells (ATCC No. CRL-1927) were plated at 10,000 cells/well in96-well tissue culture clusters (Costar) in DMEM growth medium (LifeTechnologies) supplemented with pyruvate and 10% serum (HyClone). Nextday, the medium was switched to serum free DMEM medium by substituting0.1% BSA (Fraction V; Sigma) for serum. This medium also contained theadenoviral construct KZ136 (below) that encodes a luciferase reportermini-gene driven by SRE and STAT elements, at a 1000:1 multiplicity ofinfection (m.o.i.), i.e. 1000 adenoviral particles per cell. Afterallowing 24 h for the incorporation of the adenoviral construct in thecells, the media were changed and replaced with serum-free media. Humanrecombinant IL-TIF with or without a recombinant zcytor16-Fc4 fusion wasadded at the indicated final concentration in the well (as described inTable 11, below). Dilutions of both the IL-TIF and zcytor16-Fc4 wereperformed in serum-free medium. 0.1% BSA was added for a basal assaycontrol. 4 h later, cells were lysed and luciferase activity, denotingactivation of the reporter gene, was determined in the lysate using anLuciferase Assay System assay kit (Promega) and a Labsystems Luminoskanluminometer (Labsystems, Helsinki, Finland). Activity was expressed asluciferase units (LU) in the lysate. Results are shown in Table 11,below.

TABLE 11 Level of IL-TIF (ng/ml) LU w/o zcytoR16 LU w/ 10 μg/ml zcytoR160 (basal BSA control) 103 ± 2 104 ± 2 0.03 105 ± 3 104 ± 4 0.3 108 ± 4 99 ± 6 3 134 ± 8  98 ± 15 30  188 ± 16 110 ± 3 300  258 ± 21  112 ± 30

These results demonstrate two things: First, that MES 13 cells respondto human recombinant IL-TIF and therefore possess endogenous functionalreceptors for the cytokine. Second, that the zeytoR16-Fc4 receptorfusion acts as an antagonist that effectively blocks the response toIL-TIF, even at the highest dose that this cytokine was used. Therefore,zcytor16 is an effective antagonist of IL-TIF on cells (MES 13) that areintrinsically capable of responding to IL-TIF, i.e. cells that do notrequire exogenous expression of additional receptor components torespond to the cytokine.

The construction of the adenoviral KZ136 vector was as follows. Theoriginal KZ136 vector is disclosed in Poulsen, L K et al. J. Biol. Chem.273:6228-6232, 1998. The CMV promoter/enhancer and SV40 pA sequenceswere removed from pACCMV.pLpA (T. C. Becker et al., Meth. Immunology43:161-189, 1994.) and replaced with a linker containing Asp718/KpnI andHindIII sites (oligos ZC13252 (SEQ ID NO:26) and ZC13453 (SEQ IDNO:27)). The STAT/SRE driven luciferase reporter cassette was exisedfrom vector KZ136 (Poulsen, L K et al., supra.).) as aAsp718/KpnI-HindIII fragment and inserted into the adapted pAC vector.Recombinant KZ136 Adenovirus was produced by transfection with JM17Adenovirus into 293 cells as described in T. C. Becker et al. supra.).Plaque purified virus was amplified and used to infect cultured cells at5-50 pfu/cell 12-48 hours before assay. Luciferase reporter assays wereperformed as described in 96 well microplates as per Poulsen, L K etal., supra.).

Example 17 Construct for Generating CEE-Tagged IL-TIF

Oligonucleotides were designed to generate a PCR fragment containing theKozak sequence and the coding region for IL-TIF, without its stop codon.These oligonucleotides were designed with a KpnI site at the 5′ end anda BamHI site at the 3′ end to facilitate cloning into pHZ200-CEE, ourstandard vector for mammalian expression of C-terminal Glu-Glu tagged(SEQ ID NO:10) proteins. The pHZ200 vector contains an MT-1 promoter.

PCR reactions were carried out using Turbo Pfu polymerase (Stratagene)to amplify a IL-TIF cDNA fragment. About 20 ng human IL-TIFpolynucleotide template (SEQ ID NO:14), and oligonucleotides ZC28590(SEQ ID NO:28) and ZC28580 (SEQ ID NO:29) were used in the PCR reaction.PCR reaction conditions were as follows: 95° C. for 5 minutes,; 30cycles of 95° C. for 60 seconds, 55° C. for 60 seconds, and 72° C. for60 seconds; and 72° C. for 10 minutes; followed by a 4° C. hold. PCRproducts were separated by agarose gel electrophoresis and purifiedusing a QiaQuick™ (Qiagen) gel extraction kit. The isolated,approximately 600 bp, DNA fragment was digested with KpnI and BamHI(Boerhinger-Mannheim), gel purified as above and ligated into pHZ200-CEEthat was previously digested with KpnI and BamHI.

About one microliter of the ligation reaction was electroporated intoDH10B ElectroMax™ competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werepicked and screened by PCR using oligonucleotides ZC28,590 (SEQ IDNO:28) and ZC28,580 (SEQ ID NO:29), with PCR conditions as describedabove. Clones containing inserts were then sequenced to confirmerror-free IL-TIF inserts. Maxipreps of the correct pHZ200-IL-TIF-CEEconstruct, as verified by sequence analysis, were performed.

Example 18 Transfection and Expression of IL-TIF Soluble ReceptorPolypeptides

BHK 570 cells (ATCC No. CRL-10314), were plated at about 1.2×10⁶cells/well (6-well plate) in 800 μl of serum free (SF) DMEM media (DMEM,Gibco/BRL High Glucose) (Gibco BRL, Gaithersburg, Md.). The cells weretransfected with an expression plasmid containing IL-TIF-CEE describedabove (Example 17), using Lipofectin™ (Gibco BRL), in serum free (SF)DMEM according to manufacturer's instructions.

The cells were incubated at 37° C. for approximately five hours, thentransferred to separate 150 mm MAXI plates in a final volume of 30 mlDMEM/5% fetal bovine serum (FBS) (Hyclone, Logan, Utah). The plates wereincubated at 37° C., 5% CO₂, overnight and the DNA: Lipofectin™ mixturewas replaced with selection media (5% FBS/DMEM with 1 μM methotrexate(MTX)) the next day.

Approximately 10-12 days post-transfection, colonies were mechanicallypicked to 12-well plates in one ml of 5% FCS/DMEM with 5 μM MTX, thengrown to confluence. Positive expressing clonal colonies Conditionedmedia samples were then tested for expression levels via SDS-PAGE andWestern analysis. A high-expressing clone was picked and expanded forample generation of conditioned media for purification of the IL-TIF-CEEexpressed by the cells (Example 19).

Example 19 Purification of IL-TIF Soluble Receptors from BHK 570 Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying IL-TIF polypeptide containingC-terminal GluGlu (EE) tags (SEQ ID NO:10). Conditioned media from BHKcells expressing IL-TIF-CEE (Example 18) was concentrated with an AmiconS10Y3 spiral cartridge on a ProFlux A30. A Protease inhibitor solutionwas added to the concentrated conditioned media to final concentrationsof 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). Samples were removed for analysis and the bulkvolume was frozen at −80° C. until the purification was started. Totaltarget protein concentrations of the concentrated conditioned media weredetermined via SDS-PAGE and Western blot analysis with the anti-EE HRPconjugated antibody.

About 100 ml column of anti-EE G-SEPHAROSE® (prepared as describedbelow) was poured in a Waters AP-5, 5 cm×10 cm glass column. The columnwas flow packed and equilibrated on a BioCad Sprint (PerSeptiveBioSystems, Framingham, Mass.) with phosphate buffered saline (PBS) pH7.4. The concentrated conditioned media was thawed, 0.2 micron sterilefiltered, pH adjusted to 7.4, then loaded on the column overnight withabout 1 ml/minute flow rate. The column was washed with 10 columnvolumes (CVs) of phosphate buffered saline (PBS, pH 7.4), then plugeluted with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has thesequence EYMPME (SEQ ID NO:10). The column was washed for 10 CVs withPBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of theglycine-eluted column was adjusted to 7.0 with 2 CVs of 5×PBS, thenequilibrated in PBS (pH 7.4). Five ml fractions were collected over theentire elution chromatography and absorbance at 280 and 215 nM weremonitored; the pass through and wash pools were also saved and analyzed.The EE-polypeptide elution peak fractions were analyzed for the targetprotein via SDS-PAGE Silver staining and Western Blotting with theanti-EE HRP conjugated antibody. The polypeptide elution fractions ofinterest were pooled and concentrated from 60 ml to 5.0 ml using a10,000 Dalton molecular weight cutoff membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate IL-TIF-CEE from other co-purifying proteins, theconcentrated polypeptide elution pooled fractions were subjected to aPOROS HQ-50 (strong anion exchange resin from PerSeptive BioSystems,Framingham, Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flowpacked on a BioCad Sprint. The column was counter ion charged thenequibrated in 20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). Thesample was diluted 1:13 (to reduce the ionic strength of PBS) thenloaded on the Poros HQ column at 5 ml/minute. The column was washed for10 CVs with 20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20 mMTris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions werecollected over the entire chromatography and absorbance at 280 and 215nM were monitored. The elution peak fractions were analyzed via SDS-PAGESilver staining. Fractions of interest were pooled and concentrated to1.5-2 ml using a 10,000 Dalton molecular weight cutoff membrane spinconcentrator (Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate IL-TIF-CEE polypeptide from free EE peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to size exclusion chromatography on a 1.5×90 cm SephadexS200 (Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBSat a flow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractionswere collected across the entire chromatography and the absorbance at280 and 215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified IL-TIF-CEE polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox(Sterogene) column to remove any remaining endotoxins. The sample waspassed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, theIL-TIF-CEE polypeptide was one major band. The protein concentration ofthe purified material was performed by BCA analysis (Pierce, Rockford,Ill.) and the protein was aliquoted, and stored at −80° C. according tostandard procedures.

To prepare anti-EE SBPHAROSE®, a 100 ml bed volume of proteinG-SEPHAROSE® (Pharmacia, Piscataway, N.J.) was washed 3 times with 100ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene 0.45micron filter unit. The gel was washed with 6.0 volumes of 200 mMtriethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an equalvolume of EE antibody solution containing 900 mg of antibody was added.After an overnight incubation at 4° C., unbound antibody was removed bywashing the resin with 5 volumes of 200 mM TEA as described above. Theresin was resuspended in 2 volumes of TEA, transferred to a suitablecontainer, and dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.)dissolved in TEA, was added to a final concentration of 36 mg/ml ofprotein G-SEPHAROSE® gel. The gel was rocked at room temperature for 45mm and the liquid was removed using the filter unit as described above.Nonspecific sites on the gel were then blocked by incubating for 10 mm.at room temperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA.The gel was then washed with 5 volumes of PBS containing 0.02% sodiumazide and stored in this solution at 4° C.

Example 20 Human zcytor11 Tissue Distribution in Tissue Panels UsingNorthern Blot and PCR

A. Human zcytor11 Tissue Distribution in Tissue Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor11 expressionusing PCR. The panel was made in-house and contained 94 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines are shown in Table 6 above. Aside from the PCR reaction, themethod used was as shown in Example 12. The PCR reactions were set upusing oligos ZC14,666 (SEQ ID NO: 32) and ZC14,742 (SEQ ID NO:33),Advantage 2 cDNA polymerase mix (Clontech, Palo Alto, Calif.), andRediload dye (Research Genetics, Inc., Huntsville, Ala.). Theamplification was carried out as follows: 1 cycle at 94° C. for 2minutes, 40 cycles of 94° C. for 15 seconds, 51° C. for 30 seconds and72° C. for 30 seconds, followed by 1 cycle at 72° C. for 7 minutes. Thecorrect predicted DNA fragment size was observed in bladder, brain,cervix, colon, fetal brain, fetal heart, fetal kidney, fetal liver,fetal lung, fetal skin, heart, kidney, liver, lung, melanoma, ovary,pancreas, placenta, prostate, rectum, salivary gland, small intestine,testis, thymus, trachea, spinal cord, thyroid, lung tumor, ovariantumor, rectal tumor, and stomach tumor. Zcytor11 expression was notobserved in the other tissues and cell lines tested in this panel.

A commercial 1st strand cDNA panel (Human Blood Fractions MTC Panel,Clontech, Palo Alto, Calif.) was also assayed as above. The panelcontained the following samples: mononuclear cells, activatedmononuclear cells, resting CD4+ cells, activated CD4+ cells, restingCD8+ cells, activated CD8+ cells, resting CD14+ cells, resting CD19+cells and activated CD19+ cells. All samples except activated CD8+ andActivated CD 19+ showed expression of zcytor11.

B. Tissue Distribution of Zcytor11 in Human Cell Line and Tissue PanelsUsing RT-PCR

A panel of RNAs from human cell lines was screened for zcytor11expression using RT-PCR. The panels were made in house and contained 84RNAs from various normal and cancerous human tissues and cell lines asshown in Tables 7-10 above. The RNAs were made from in house orpurchased tissues and cell lines using the RNAeasy Midi or Mini Kit(Qiagen, Valencia, Calif.). The panel was set up in a 96-well formatwith 100 ngs of RNA per sample. The RT-PCR reactions were set up usingoligos ZC14,666 (SEQ ID NO:32) and ZC14,742 (SEQ ID NO:33), Rediload dyeand SUPERSCRIPT One Step RT-PCR System (Life Technologies, Gaithersburg,Md.). The amplification was carried out as follows: one cycle at 50° for30 minutes followed by 45 cycles of 94°, 15 seconds; 52°, 30 seconds;72°, 30 seconds; then ended with a final extension at 72° for 7 minutes.8 to 10 uls of the PCR reaction product was subjected to standardAgarose gel electrophoresis using a 4% agarose gel. The correctpredicted cDNA fragment size was observed in adrenal gland, bladder,breast, bronchus, normal colon, colon cancer, duodenum, endometrium,esophagus, gastic cancer, gastro-esophageal cancer, heart ventricle,ileum, normal kidney, kidney cancer, liver, lung, lymph node, pancreas,parotid, skin, small bowel, stomach, thyroid, and uterus. Cell linesshowing expression of zcytor11 were A-431, differentiated CaCO2, DLD-1,HBL-100, HCT-15, HepG2, HepG2+IL6, HuH7, and NHEK #1-4. Zcytor11expression was not observed in the other tissues and cell lines testedin this panel.

In addition, because the expression pattern of zcytor11, one of IL-TIF'sreceptors, shows expression in certain specific tissues, bindingpartners including the natural ligand, IL-TIF, can also be used as adiagnostic to detect specific tissues (normal or abnormal), cancer, orcancer tissue in a biopsy, tissue, or histologic sample, particularly intissues where IL-TIF receptors are expressed. IL-TIF can also be used totarget other tissues wherein its receptors, e.g., zcytor16 and zcytor11are expressed. Moreover, such binding partners could be conjugated tochemotherapeutic agents, toxic moieties and the like to target therapyto the site of a tumor or diseased tissue. Such diagnostic and targetedtherapy uses are known in the art and described herein.

The expression patterns of zcytor11 (above) and zcytor16 (Example 12,and Example 21) indicated target tissues and cell types for the actionof IL-TIF, and hence IL-TIF antagonsists, such as zcytor16. The zcytor11expression generally overlapped with zcytor16 expression in threephysiologic systems: digestive system, female reproductive system, andimmune system. Moreover, the expression pattern of the receptor(zcytor11) indicated that an IL-TIF antagonist such as zcytor16 wouldhave therapeutic application for human disease in two areas:inflammation (e.g., IBD, Chron's disease, pancreatitis) and cancer(e.g., ovary, colon). That is, the polynucleotides, polypeptides andantibodies of the present invention can be used to antagonize theinflammatory, and other cytokine-induced effects of IL-TIF interactionwith the cells expressing the zcytor11 receptor.

Moreover, the expression of zcytor11 appeared to be downregulated orabsent in an ulcerative colitis tissue, HepG2 liver cell line induced byIL-6, activated CD8+ T-cells and CD19+B-cells. However, zcytor16appeared to be upregulated in activated CD19+B-cells (Example 12), whilezcytor11 is downregulated in activated CD19+ cells, as compared to theresting CD19+ cells (above). The expression of zcytor11 and zcytor16 hasa reciprocal correlation in this case. These RT-PCR experimentsdemonstrate that CD19+ peripheral blood cells, B lymphocytes, expressreceptors for IL-TIF, namely zcytoR11 and zcytoR16. Furthermore B cellsdisplay regulated expression of zcytoR11 and zcytoR16. B-lymphocytesactivated with mitogens decrease expression of zcytoR11 and increaseexpression of zcytoR16. This represents a classical feedback inhibitionthat would serve to dampen the activity of IL-TIF on B cells and othercells as well. Soluble zcytoR16 would act as an antagonist to neutralizethe effects of IL-TIF on B cells. This would be beneficial in diseaseswhere B cells are the key players: Autoimmune diseases includingsystemic lupus erythmatosus (SLE), myasthenia gravis, immune complexdisease, and B-cell cancers that are exacerbated by IL-TIF. Alsoautoimmune diseases where B cells contribute to the disease pathologywould be targets for zcytoR16 therapy: Multiple sclerosis, inflammatorybowel disease (IBD) and rheumatoid arthritis are examples. ZcytoR16therapy would be beneficial to dampen or inhibit B cells producing IgEin atopic diseases including asthma, allergy and atopic dermatitis wherethe production of IgE contributes to the pathogenesis of disease.

B cell malignancies may exhibit a loss of the “feedback inhibition”described above. Administration of zcytoR16 would restore control ofIL-TIF signaling and inhibit B cell tumor growth. The administration ofzcytoR16 following surgical resection or chemotherapy may be useful totreat minimal residual disease in patients with B cell malignancies. Theloss of regulation may lead to sustain or increased expression ofzcytoR11. Thus creating a target for therapeutic monoclonal antibodiestargeting zcytoR11.

Example 21 Identification of Cells Expressing zcytor16 Using In SituHybridization

Specific human tissues were isolated and screened for zcytor16expression by in situ hybridization. Various human tissues prepared,sectioned and subjected to in situ hybridization included cartilage,colon, appendix, intestine, fetal liver, lung, lymph node, lymphoma,ovary, pancreas, placenta, prostate, skin, spleen, and thymus. Thetissues were fixed in 10% buffered formalin and blocked in paraffinusing standard techniques. Tissues were sectioned at 4 to 8 microns.Tissues were prepared using a standard protocol (“Development ofnon-isotopic in situ hybridization” at The Laboratory of ExperimentalPathology (LEP), NIEHS, Research Triangle Park, NC. Briefly, tissuesections were deparaffinized with HistoClear (National Diagnostics,Atlanta, Ga.) and then dehydrated with ethanol. Next they were digestedwith Proteinase K (50 μg/ml) (Boehringer Diagnostics, Indianapolis, Id.)at 37° C for 2 to 7 minutes. This step was followed by acetylation andre-hydration of the tissues.

One in situ probe was designed against the human zcytor16 sequence(nucleotide 1-693 of SEQ ID NO:1), and isolated from a plasmidcontaining SEQ ID NO:1 using standard methods. T3 RNA polymerase wasused to generate an antisense probe. The probe was labeled withdigoxigenin (Boehringer) using an In Vitro transcription System(Promega, Madison, Wis.) as per manufacturer's instruction.

In situ hybridization was performed with a digoxigenin-labeled zcytor16probe (above). The probe was added to the slides at a concentration of 1to 5 pmol/ml for 12 to 16 hours at 62.5° C. Slides were subsequentlywashed in 2×SSC and 0.1×SSC at 55° C. The signals were amplified usingtyramide signal amplification (TSA) (TSA, in situ indirect kit; NEN) andvisualized with VECTOR RED™ substrate kit (Vector Lab) as permanufacturer's instructions. The slides were then counter-stained withhematoxylin (Vector Laboratories, Burlingame, Calif.).

Signals were observed in several tissues tested: The lymph node, plasmacells and other mononuclear cells in peripheral tissues were stronglypositive. Most cells in the lymphatic nodule were negative. In lymphomasamples, positive signals were seen in the mitotic and multinuclearcells. In spleen, positive signals were seen in scattered mononuclearcells at the periphery of follicles were positive. In thymus, positivesignals were seen in scattered mononuclear cells in both cortex andmedulla were positive. In fetal liver, a strong signal was observed in amixed population of mononuclear cells in sinusoid spaces. A subset ofhepatocytes might also have been positive. In the inflamed appendix,mononuclear cells in peyer's patch and infiltration sites were positive.In intestine, some plasma cells and ganglia nerve cells were positive.In normal lung, zcytor16 was expressed in alveolar epithelium andmononuclear cells in interstitial tissue and circulation. In the lungcarcinoma tissue, a strong signal was observed in mostly plasma cellsand some other mononuclear cells in peripheral of lymphatic aggregates.In ovary carcinoma, epithelium cells were strongly positive. Someinterstitial cells, most likely the mononuclear cells, were alsopositive. There was no signal observed in the normal ovary. In bothnormal and pancreatitis pancreas samples, acinar cells and somemononuclear cells in the mesentery were positive. In the early term (8weeks) placenta, signal was observed in trophoblasts. In skin, somemononuclear cells in the inflamed infiltrates in the superficial dermiswere positive. Keratinocytes were also weakly positive. In prostatecarcinoma, scatted mononuclear cells in interstitial tissues werepositive. In articular cartilage, chondrocytes were positive. Othertissues tested including normal ovary and a colon adenocarcinoma werenegative.

In summary, the in situ data was consistent with expression datadescribed above for the zcytor16. Zcytor16 expression was observedpredominately in mononuclear cells, and a subset of epithelium was alsopositive. These results confirmed the presence of zcytor16 expression inimmune cells and point toward a role in inflammation, autoimmunedisease, or other immune function, for example, in bindingpro-inflammatory cytokines, including but not limited to IL-TIF.Moreover, detection of zcytor16 expression can be used for example as anmarker for mononuclear cells in histologic samples.

Zcytor16 is expressed in mononuclear cells, including normal tissues(lymph nodes, spleen, thymus, pancreas and fetal liver, lung), andabnormal tissues (inflamed appendix, lung carcinoma, ovary carcinoma,pancreatitis, inflamed skin, and prostate carcinoma). It is notable thatplasma cells in the lymph node, intestine, and lung carcinoma arepositive for zcytor16. Plasma cells are immunologically activatedlymphocytes responsible for antibody synthesis. In addition, IL-TIF, isexpressed in activated T cells. In addition, the expression of zcytor16is detected only in activated (but not in resting) CD4+ and CD19+ cells(Example 12). Thus, zcytor16 can be used as a marker for or as a targetin isolating certain lymphocytes, such as mononuclear leucocytes andlimited type of activated leucocytes, such as activated CD4+ and CD19+.

Furthermore, the presence of zcytor16 expression in activated immunecells such as activated CD4+ and CD19+ cells showed that zcytor16 may beinvolved in the body's immune defensive reactions against foreigninvaders: such as microorganisms and cell debris, and could play a rolein immune responses during inflammation and cancer formation.

Moreover, as discussed herein, epithelium form several tissues waspositive for zcytor16 expression, such as hepatocytes (endoderm-derivedepithelia), lung alveolar epithelium (endoderm-derived epithelia), andovary carcinoma epithelium (mesoderm-derived epithelium). The epitheliumexpression of zeytor16 could be altered in inflammatory responses and/orcancerous states in liver and lung. Thus, Zcytor16 could be used asmarker to monitor changes in these tissues as a result of inflammationor cancer. Moreover, analysis of zcytor16 in situ expression showed thatnormal ovary epithelium is negative for zcytor16 expression, while it isstrongly positive in ovary carcinoma epithelium providing furtherevidence that zcytor16 polynucleotides, polypeptides and antibodies canbe used as a diagnostic marker and/or therapeutic target for thediagnosis and treatment of ovarian cancers, and ovary carcinoma, asdescribed herein.

Zcytor16 was also detected in other tissues, such as acinar cells inpancreas (normal and pancreatitis tissues), trophoblasts in placenta(ectoderm-derived), chondrocytes in cartilage (mesoderm-derived), andganglia cells in intestine (ectoderm-derived). As such, zcytor16 may beinvolved in differentiation and/or normal functions of correspondingcells in these organs. As such, potential utilities of zcytor16 includemaintenance of normal metabolism and pregnancy, boneformation/homeostasis, and physiological function of intestine, and thelike.

Example 22 In Vivo Affects of IL-TIF Polypeptide

Mice (female, C57B1, 8 weeks old; Charles River Labs, Kingston, N.Y.)were divided into three groups. An adenovirus expressing an IL-TIFpolypeptide (# SEQ ID NO:15) was previously made using standard methods.On day 0, parental or IL-TIF adenovirus was administered to the first(n=8) and second (n=8) groups, respectively, via the tail vein, witheach mouse receiving a dose of ˜1×10¹¹ particles in ˜0.1 ml volume. Thethird group (n=8) received no treatment. On days 12, mice were weighedand blood was drawn from the mice. Samples were analyzed for completeblood count (CBC) and serum chemistry. Statistically significantelevations in neutrophil and platelet counts were detected in the bloodsamples from the IL-TIF adenovirus administered group relative to theparental adenovirus treated group. Also, lymphocyte and red blood cellcounts were significantly reduced from the IL-TIF adenovirusadministered group relative to the parental adenovirus treated group. Inaddition, the IL-TIF adenovirus treated mice decreased in body weight,while parental adenovirus treated mice gained weight.

The results suggested that IL-TIF affects hematopoiesis, i.e., bloodcell formation in vivo. As such, IL-TIF could have biological activitieseffecting different blood stem cells, thus resulting increase ordecrease of certain differentiated blood cells in a specific lineage.For instance, IL-TIF appears to reduce lymphocytes, which is likely dueto inhibition of the committed progenitor cells that give rise tolymphoid cells. IL-TIF also decreases red blood cells. This findingagrees with the inhibitory effects of IL-TIF on the proliferation and/orgrowth of myeloid stem cells (Example 23), supporting the notion thatIL-TIF could play a role in anemia, infection, inflammation, and/orimmune diseases by influencing blood cells involved in these process.Antagnists against IL-TIF, such as antibodies or its soluble receptorzcytor16, could be used as therapeutic reagents in these diseases.

Moreover, these experiments using IL-TIF adenovirus in mice suggest thatIL-TIF over-expression increases the level of neutrophils and plateletsin vivo. Although this may appear contradictory to the finding seen inK562 cells (Example 23), it is not uncommon to observe diverseactivities of a particular protein in vitro versus in vivo. It isconceivable that there are other factors (such as cytokines and modifiergenes) involved in the responses to IL-TIF in the whole animal system.Nevertheless, these data strongly support the involvement of IL-TIF inhematopoiesis. Thus, IL-TIF and its receptors are suitablereagents/targets for the diagnosis and treatment in variety ofdisorders, such as inflammation, immune disorders, infection, anemia,hematopoietic and other cancers, and the like.

Example 23 The IL-TIF Polypeptide Lyses K-562 Cells in CytotoxicityAssay

The K-562 cell line (CRL-243, ATCC) has attained widespread use as ahighly sensitive in vitro target for cytotoxicity assays. K-562 blastsare multipotential, hematopoietic malignant cells that spontaneouslydifferentiate into recognizable progenitors of the erythrocytic,granulocytic and monocytic series (Lozzio, B B et al., Proc. Soc. Exp.Biol. Med. 166: 546-550, 1981).

K562 cells were plated at 5,000 cells/well in 96-well tissue cultureclusters (Costar) in DMEM phenol-free growth medium (Life Technologies)supplemented with pyruvate and 10% serum (HyClone). Next day, humanrecombinant IL-TIF (Example 19), BSA control or retinoic acid (known tobe cytotoxic to K562 cells) were added. Seventy-two hours later, thevital stain MTT (Sigma, St Louis, Mo.), a widely used indicator ofmitochondrial activity and cell growth, was added to the cells at afinal concentration of 0.5 mg/ml. MMP is converted to a purple formazanderivative by mitochondrial dehydrogenases. Four hours later, convertedMMP was solubilized by adding an equal volume of acidic isopropanol(0.04N HC1 in absolute isopropanol) to the wells. Absorbance wasmeasured at 570 nm, with background subtraction at 650 nm. In thisexperimental setting, absorbance reflects cell viability. Results shownin Table 12 are expressed as % cytotoxicity.

TABLE 12 Agent Concentration % Cytotoxicity BSA Control 1 ug/ml 1.3Retinoic acid 100 uM 62 IL-TIF 100 ng/ml 16.2 IL-TIF 300 ng/ml 32

The results indicate that IL-TIF may affect myeloid stem cells. Myeloidstem cells are daughter cells of the universal blood stem cells. Theyare progenitors of erythrocytes, platelets megakaryocytes, monocytes (ormigrated macrophages), neutrophil and basophil, etc. Since K-562 blastsspontaneously differentiate into progenitors of the erythrocytic,granulocytic and monocytic series, it can be considered as myeloid stemcells. Thus, the results demonstrate that IL-TIF has an inhibitoryactivity on the proliferation and/or growth of myeloid stem cells. ThusIL-TIF could play a role in anemia, infection, inflammation, and/orimmune diseases. In addition, an antaganist against IL-TIF, such asantibodies or its soluble receptor zcytor16, could be used to blockIL-TIF's activity on myeloid stem cells, or as therapeutic reagents indiseases such as anemia, infection, inflammation, and/or immunediseases.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for inhibiting T-cell inducible factor (IL-TIF) inducedproliferation of hematopoietic cells and hematopoietic cell progenitorscomprising culturing in the presence of IL-TIF bone marrow or peripheralblood cells with a composition comprising a soluble cytokine receptorcomprising amino acid residues 22-231 or 22-210 of SEQ ID NO:2, whereinproliferation of the hematopoietic cells in the bone marrow orperipheral blood cells is reduced as compared to bone marrow orperipheral blood cells cultured in the absence of the soluble cytokinereceptor.
 2. The method of claim 1, wherein the hematopoietic cells andhematopoietic progenitor cells are lymphoid cells.
 3. The method ofclaim 2, wherein the lymphoid cells are macrophages or T cells.